Rapid enumeration of Escherichia coli in marine bathing waters: potential interference of nontarget bacteria

Aims: To compare the Escherichia coli quantification given by the ‘Coliplage^®’ assay, based on the direct measurement of the β‐d ‐glucuronidase (GLUase) activity and the reference Most Probable Number (MPN) method from seawater sites and investigate the possible interference of non‐E. coli strains in the GLUase activity measurement. Methods and Results: Comparison performed from 69 French coastal bathing sites (1401 samples) showed nonconcordance between both methods, only for 8% of samples. Non‐E. coli 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG+) were isolated from nonconcordant samples. Phylogenetic analysis showed that Gammaproteobacteria were dominants and mainly represented by Vibrio species, which displayed GLUase activities on the same order of magnitude and sometimes much higher as E. coli reference strains. Conclusions: The‘Coliplage^®’ assay is a rapid method for the quantification of E. coli showed few discordances with the standard MPN method. Some Vibrio species could interfere on the direct GLUase activity measurement of E. coli. Significance and Impact of the Study: Data present the first qualitative investigation on disagreement between Coliplage^® and the MPN results. If the interference of Vibrio species is confirmed in situ, appropriate treatments should be developed to remove the interfering signal.     

Evaluation of a new medium for the enumeration of total coliforms and Escherichia coli in Japanese surface waters

Aim: A new medium, EC‐Blue‐10, containing chromogenic and fluorogenic substrates, KNO₃ and sodium pyruvate has been developed for the rapid simultaneous detection and enumeration of total coliforms and Escherichia coli in water. Methods and Results:  Two evaluations of EC‐Blue‐10 were carried out. Firstly, EC‐Blue‐10 was compared with Colilert‐MPN for 96 water samples using MPN for total coliforms and E. coli. Secondly, the detection of coliforms and E. coli were compared using 2400 tubes of EC‐Blue‐10 and Colilert‐MPN. The regression coefficients between EC‐Blue‐10 and Colilert‐MPN for total coliforms and E. coli were 0·91 and 0·89, respectively. For the detection results, the Cohen’s kappa values between the two media were 0·79 for coliforms and 0·72 for E. coli. Conclusions: EC‐Blue‐10 is almost same as Colilert‐MPN for the detection of coliforms and E. coli in surface waters. Further evaluation for EC‐Blue‐10 is needed to verify in different geographical areas. Significance and Impact of the Study: EC‐Blue‐10 is useful method for the rapid and simultaneous detection of total coliforms and E. coli in water sample.     

The effect of the native bacterial community structure on the predictability of E. coli O157:H7 survival in manure‐amended soil

Aims: The survival capability of pathogens like Escherichia coli O157:H7 in manure‐amended soil is considered to be an important factor for the likelihood of crop contamination. The aim of this study was to reveal the effects of the diversity and composition of soil bacterial community structure on the survival time (ttd) and stability (irregularity, defined as the intensity of irregular dynamic changes in a population over time) of an introduced E. coli O157:H7 gfp‐strain were investigated for 36 different soils by means of bacterial PCR‐DGGE fingerprints. Methods and Results: Bacterial PCR‐DGGE fingerprints made with DNA extracts from the different soils using bacterial 16S‐rRNA‐gene‐based primers were grouped by cluster analysis into two clusters consisting of six and 29 soils and one single soil at a cross‐correlation level of 16% among samples per cluster. Average irregularity values for E. coli O157:H7 survival in the same soils differed significantly between clusters (P = 0·05), whereas no significant difference was found for the corresponding average ttd values (P = 0·20). The irregularity was higher for cluster 1, which consisted primarily of soils that had received liquid manure and artificial fertilizer and had a significant higher bacterial diversity and evenness values (P < 0·001). Conclusions: Bacterial PCR‐DGGE fingerprints of 36 manure‐amended soils revealed two clusters which differed significantly in the stability (irregularity) of E. coli O157 decline. The cluster with the higher irregularity was characterized by higher bacterial diversity and evenness. Significance and Impact of the Study: The consequence of a high temporal irregularity is a lower accuracy of predictions of population behaviour, which results in higher levels of uncertainty associated with the estimates of model parameters when modelling the behaviour of E. coli O157:H7 in the framework of risk assessments. Soil community structure parameters like species diversity and evenness can be indicative for the reliability of predictive models describing the fate of pathogens in (agricultural) soil ecosystems.     

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil*

Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4‐week‐old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant^?1 and 1 ml in soil, resulting in 6·5 log CFU 200 g^?1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co‐inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa. Methods and Results: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g^?1 in manure and manure‐amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro‐Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high‐density inocula and enhanced the decline of low‐density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure‐amended soil than in manure. The 7 log CFU g^?1E. coli O157:H7 and Salm. Typhimurium survived for 49–84 and 63–98 days, while at 4 log CFU g^?1, persistence was 21–28 and 35–42 days, respectively. Conclusions: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. Significance and Impact of the Study: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.     

Serogroup distribution and virulence characteristics of sorbitol‐negative Escherichia coli from food and cattle stool

Aims: To (i) study the serogroup distribution and virulence characteristics of non‐sorbitol‐fermenting Escherichia coli isolates from foods of animal origin and cattle faeces and (ii) re‐examine the true sorbitol and β‐d ‐glucuronidase (GUD) reactions of sorbitol‐negative (Sor^?) strains from MacConkey sorbitol agar (SMAC) to assess their phenotypic similarity with E. coli O157. Methods and Results: One hundred and thirty Sor^?E. coli were isolated from 556 food samples and 177 cattle stool samples using cefixime tellurite–supplemented SMAC (CT‐SMAC) and chromogenic HiCrome MS.O157 agar respectively. Based on typing of somatic antigen, the isolates were classified into 38 serogroups. PCR results identified about 40% strains, belonging to O5, O8, O20, O28, O48, O60, O78, O82, O84, O101, O110, O123, O132, O156, O157, O‐rough and OUT as Shiga toxigenic. Majority of O5, O84, O101, O105, O123, O157, O‐rough and OUT strains were enterohaemolytic. Further, 39·2% and 63·1% of Sor^? isolates from CT‐SMAC fermented sorbitol in phenol red broth and hydrolysed 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG) respectively. Members of serogroups O5, O28, O32, O81, O82, O84, O101, O‐rough lacked both the sorbitol fermentation (broth test) and GUD activity and might create confusion in phenotypic identification of E. coli O157. Conclusions: Sor^?E. coli isolates from raw meat, milk, shrimp and cattle stool belonged to 38 serogroups, with E. coli O157 constituting only 14·6% of the isolates. Many of these nonclinical Sor^? strains were potentially pathogenic. Nearly 39% of these Sor^?E. coli from CT‐SMAC fermented sorbitol in broth, indicating the need for confirmation of sorbitol reaction in broth. Significance and Impacts of the Study: Classical sorbitol utilization and GUD tests are not likely definitive tests for E. coli O157. Further improvement of differential media based on these phenotypic properties is necessary for detection of pathogenic serotypes from foods and environmental samples.     

Fate of Escherichia coli O145 present naturally in bovine slurry applied to vegetables before harvest,after washing and simulated wholesale and retail distribution

Aims

To determine the fate of Escherichia coli on vegetables that were processed through commercial wash treatments and stored under simulated retail conditions at 4°C or wholesale at fluctuating ambient temperatures (0–25°C, dependent on season).

Methods and Results

Bovine slurry that was naturally contaminated with E. coli O145 was applied without dilution or diluted 1:10 using borehole water to growing potatoes, leeks or carrots. Manure was applied 1 week prior to harvest to simulate a near‐harvest contamination event by manure deposition or an application of contaminated water to simulate a flooding event or irrigation from a contaminated water source. At harvest, crops were contaminated at up to 2 log cfu g^?1. Washing transferred E. coli into the water of a flotation tank used for potato washing and did not completely remove all traces of contamination from the crop. Manure‐contaminated potatoes were observed to contain 0·72 cfu E. coli O145 g^?1 after processing and retail storage. Manure‐contaminated leeks harboured 0·73–1·55 cfu E. coli O145 g^?1 after washing and storage. There was no cross‐contamination when leeks were spray washed. Washing in an abrasive drum resulted in less than perfect decontamination for manure‐contaminated carrots. There were five post‐distribution isolations from carrots irrigated with contaminated water 24 h prior to harvest.

Conclusions

Standard commercial washing and distribution conditions may be insufficient to reliably control human pathogenic E. coli on fresh produce.

Significance and Impact

Previous speculation that the cause of a UK foodborne disease outbreak was soil from imperfectly cleaned vegetables is plausible.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Comparison of TEMPO® EC and TBX medium for the enumeration of Escherichia coli in cheese

Aims: TEMPO^® EC (Escherichia coli) is based on glucuronidase activity and is a test for use with the TEMPO system for the automated 24 h enumeration of E. coli in food products. In this study, TEMPO EC was compared with TBX medium, the current standard plate method for the enumeration of E. coli in cheese. Methods and Results: For comparative purposes, both naturally (92) and artificially contaminated (31) cheese samples were studied. Pearson correlation coefficients were determined as 0.954 and 0.978 between the two methods for naturally and artificially contaminated samples, respectively. Regression analysis yielded the following equations: log₁₀ TEMPO EC = 0.340 + 0.889 log₁₀ TBX medium and log₁₀ TEMPO EC = 0.174 + 0.899 log₁₀ TBX medium for naturally and artificially contaminated samples, respectively. In general, absolute differences did not exceed one log between results obtained by the two methods. Conclusions: Statistical analysis of the results showed good agreement between the two enumeration methods. Significance and Impact of the Study: TEMPO EC is a practical and reliable alternative to the current standard plate method for the enumeration of E. coli in foods.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

Quantification of Survival of Escherichia coli O157:H7 on Plants Affected by Contaminated Irrigation Water

Enterohemorrhagic E. coli O157: H7 (EHEC) is a major foodborne pathogen capable of causing diarrhea and vomiting, with further complications such as hemolytic‐uremic syndrome (HUS). The aim of this study was to use the real‐time PCR method to quantify the survival of Escherichia coli O157:H7/pGFP in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non‐rhizosphere soils (sand and clay) irrigated with contaminated water and compare the results obtained between real‐time PCR method and conventional plate counts. The real‐time PCR probe was designed to hybridize with the (eae) gene of E. coli O157:H7. The probe was incorporated into real‐time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non‐rhizosphere soils irrigated with water artificially contaminated with E. coli O157:H7. The detection limit for E. coli O157:H7 quantification by real‐time PCR was 2.3 × 10³ in the rhizosphere and phyllosphere samples. E. coli O157:H7 survived longer in rhizosphere soil than the non‐rhizosphere soil. The concentration of E. coli O157:H7/pGFP in rhizosphere soils was ≥ 10⁴ CFU/g in both soils at day 12 based on both plate count and real time PCR, with the clay soil significantly (P = 0.05) higher than the sandy soil. This data showed that E. coli O157H:7 can persist in the environment for more than 50 d, and this may pose some risk for both animal and human infection and provides a very significant pathway for pathogen recontamination in the environment.     

Enterotoxigenic Escherichia coli in sewage‐impacted waters and aquatic weeds: quantitative PCR for culture‐independent enumeration

Aim: To develop quantitative PCR for culture‐independent enumeration of enterotoxigenic Escherichia coli (ETEC) in sewage‐impacted waters and aquatic weeds. Methods and Results: Two fluorescent probes (TaqMan and FRET) based on two different real‐time PCR chemistries were designed in highly conserved region of LT1 gene encoding heat labile enterotoxin. Both the assays could detect 2 CFU ml^?1 from serially diluted (two‐fold and ten‐fold) culture of reference strain (E. coli MTCC 723). FRET performed better in terms of C_T value and PCR efficiency than TaqMan. The presence of 10⁶ CFU ml^?1 of nonpathogenic E. coli reduced the detection limit two‐fold with both the probes. However, the performance for two chemistries in various environmental samples was significantly (student’s t‐test, P < 0·05) different. Conclusion: It could be inferred from this study that real‐time PCR chemistries (TaqMan and FRET) could detect very few copies of target DNA in pure cultures, but may give varied response in the presence of nonspecific DNA and natural inhibitors present in environmental sample matrices. Significance and Impact of the Study: The assays can be used for pre‐emptive monitoring of aquatic weeds (a potential nonpoint source), surface and potable waters to prevent waterborne outbreaks caused by ETEC.     

Interactions Between Escherchia coli and the Colloids of Three Variable Charge Soils and Their Effects on Soil Surface Charge Properties

The adhesion of Escherchia coli (E. coli) to the colloids of three variable charge soils and its effect on surface charge properties and potassium adsorption of these soil colloids were investigated. The adhesion isotherms of E. coli by soil colloids can be described using the Langmuir equation. The amount of E. coli adhered by the soil colloids varied with soil type and followed the order: Ultisol from Guangxi > Oxisol from Yunnan > Ultisol from Jiangxi. The iron and aluminum oxide contents and CECs of the soils are the important factors affecting the adhesion of E. coli to soil colloids. The relatively lower iron and aluminum oxide contents and higher CEC of the Ultisol from Jiangxi led to the lower adhesion of E. coli to the soil colloids compared to the Ultisol from Guangxi and the Oxisol from Yunnan. The amount of E. coli adhered to the soil colloids decreased with increasing pH, which was consistent with the results predicted from the DLVO theory. E. coli adhesion made the zeta potential of the soil colloids more negative and reduced the isoelectric point of the soil colloids, suggesting that E. coli decreased the surface positive charge and increased negative charge of the soil colloids. In addition, E. coli adhesion increased K⁺ adsorption by the soil colloids. Therefore, bacterial adhesion improves the fertility of variable charge soils by increasing soil CEC because the CECs of variable charge soils are usually low.     

Survival and spread of Shiga toxin‐producing Escherichia coli in alpine pasture grasslands

Aims: To determine the fate of Shiga toxin‐producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. Methods and Results: During the summers of 2005 and 2006, the field survival of STEC was monitored in cowpats and underlying soils in four different alpine pasture units. A most probable number (MPN)‐PCR stx assay was used to enumerate STEC populations. STEC levels ranged between 3·9 and 5·4 log₁₀ CFU g^?1 in fresh cowpats and slowly decreased until their complete decay (inactivation rates k < 0·04 day^?1). PFGE typing of STEC strains isolated from faecal and soil samples assessed the persistence of various clonal types for at least 2 months in cowpats and their vertical dispersal down through the soil at a depth up to at least 20 cm. STEC cells counts in soil were always below 2 log₁₀ CFU g^?1, regardless of the pasture unit investigated. The soil became rapidly free of detectable STEC once the cowpat had decomposed. The eight STEC strains isolated during this study belonged to six distinct serotypes and tested positive for the gene(s) stx2, including the stx2g and stx2 NV206 variants. Conclusions: STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. Significance and impact of the Study: This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.     

Escherichia coli Contamination of Vegetables Grown in Soils Fertilized with Noncomposted Bovine Manure: Garden-Scale Studies

In this study we tested the validity of the National Organic Program (NOP) requirement for a ≥120-day interval between application of noncomposted manure and harvesting of vegetables grown in manure-fertilized soil. Noncomposted bovine manure was applied to 9.3-m² plots at three Wisconsin sites (loamy sand, silt loam, and silty clay loam) prior to spring and summer planting of carrots, radishes, and lettuce. Soil and washed (30 s under running tap water) vegetables were analyzed for indigenous Escherichia coli. Within 90 days, the level of E. coli in manure-fertilized soil generally decreased by about 3 log CFU/g from initial levels of 4.2 to 4.4 log CFU/g. Low levels of E. coli generally persisted in manure-fertilized soil for more than 100 days and were detected in enriched soil from all three sites 132 to 168 days after manure application. For carrots and lettuce, at least one enrichment-negative sample was obtained ≤100 days after manure application for 63 and 88% of the treatments, respectively. The current ≥120-day limit provided an even greater likelihood of not detecting E. coli on carrots (≥1 enrichment-negative result for 100% of the treatments). The rapid maturation of radishes prevented conclusive evaluation of a 100- or 120-day application-to-harvest interval. The absolute absence of E. coli from vegetables harvested from manure-fertilized Wisconsin soils may not be ensured solely by adherence to the NOP ≥120-day limit. Unless pathogens are far better at colonizing vegetables than indigenous E. coli strains are, it appears that the risk of contamination for vegetables grown in Wisconsin soils would be elevated only slightly by reducing the NOP requirement to ≥100 days.     

Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

Effect of biofumigation with manure amendments and repeated biosolarization on <Emphasis Type="Italic">Fusarium</Emphasis> densities in pepper crops

In the region of Murcia (southeast Spain), sweet pepper has been grown as a monoculture in greenhouses for many years. Until 2005, when it was banned, soils were disinfested with methyl bromide (MB) to control pathogens and to prevent soil fatigue effects. The genus Fusarium plays an important role in the microbiological component associated with yield decline in pepper monocultures. In the present study, soils were treated with manure amendments, alone (biofumigation, B) or in combination with solarization (biosolarization, BS), with or without the addition of pepper plant residues. The B and BS treatments were compared with a treatment using MB. The extent of disinfestation was measured from the density of Fusarium spp. isolated from the soil before and after the respective treatments. Three different species were systematically isolated: Fusarium oxysporum, Fusarium solani and Fusarium equiseti. The repeated use of manure amendments with pepper crop residues, without solarization, was unable to decrease the Fusarium spp. density (which increased from 2,047.17 CFU g⁻¹ to 3,157.24 CFU g⁻¹ before and after soil disinfestation, respectively), unlike MB-treated soil (in which the fungi decreased from 481.39 CFU g⁻¹ to 23.98 CFU g⁻¹). However, the effectiveness of the repeated application of BS in diminishing doses (with or without adding plant residues) on Fusarium populations (reductions greater than 72%) was similar to or even greater than the effect of MB.     

Expansion of the ω‐oxidation system AlkBGTL of Pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono‐esterified dicarboxylic acid production in E. coli

The AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U g_cdw^?1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol^?1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.     

Occurrence of thermotolerant Campylobacter species in surface waters of a Mediterranean area and in its prevailing pollution sources

Aims: To determine the prevalence of Campylobacter in surface waters of a highly populated Mediterranean area. Methods and Results: Surface water and wastewater samples were collected from an area in the north‐east of Spain during a 2‐year study. All the samples were analysed using the MPN method and Multiplex PCR to quantify and identify Campylobacter. It was detected in 82% of the samples from the Llobregat River with a mean of 1·3 MPN 100 ml^?1. The lowest counts were obtained in summer. Campylobacter coli was the predominant species in this river. The bacteria were isolated from marsh water but not from seawater samples. The highest counts of campylobacters were found in poultry wastewater where Camp. jejuni was the predominant species, as in urban sewage. In pig slurry, Camp. coli was the only species detected. Conclusions: Campylobacter jejuni and Camp. coli are present and widely distributed in the surface water of the studied area. The two species co‐exist, with Camp. coli being predominant. In river water, campylobacter counts presented a seasonal distribution. No relationship with faecal indicators was found. Significance and Impact of the Study: This study provides the first data on the occurrence and concentrations of thermotolerant campylobacter species in surface water in a Mediterranean area.