The occurrence of enteric pathogens and Aeromonas species in organic vegetables

International Journal of Food Microbiology 70 Ž2001. 155–162 www.elsevier.comrlocaterijfoodmicro

The occurrence of enteric pathogens and Aeromonas species in organic vegetables M.A.S. McMahon a,) , I.G. Wilson b a

Food Microbiology Research Group, Room 15 G 15, UniÕersity of Ulster, Shore Road, Newtownabbey, Co Antrim., BT37 0QB, N. Ireland, UK b Northern Ireland Public Health Laboratory, Belfast City Hospital, Lisburn Road, Belfast, BT9 7AD, N. Ireland, UK Received 17 November 2000; received in revised form 20 March 2001; accepted 2 May 2001

Abstract A range of commercially available organic vegetables Ž n s 86. was examined for the presence of Salmonella, Campylobacter, Escherichia coli, E. coli O 157, Listeria and Aeromonas spp., to provide information on the occurrence of such organisms in organic vegetables in Northern Ireland. The study was not designed to quantify such organisms or to compare occurrence with conventionally farmed vegetables. Standard enrichment techniques were used to isolate and identify enteric pathogens and Aeromonas species. No Salmonella, Campylobacter, E. coli, E. coli O 157, Listeria were found in any of the samples examined. Aeromonas species were isolated from 34% of the total number of organic vegetables examined. Many Ž64%. of the organic vegetables examined were Aready-to-eatB after minimal processing, i.e., washing. Aeromonas spp. was isolated from 41% of these vegetables. Aeromonas spp. was not recovered from certain vegetable types. The most commonly isolated species of Aeromonas was Aeromonas schubertii with 21.0% of all samples contaminated with this species; 5.8% of samples contained A. hydrophila, 5.8% A. trota, 3.5% A. caÕiae and 2.3% contained A. Õeronii biovar Õeronii. Although Aeromonas species are frequently detected in organic vegetables, the absence of accepted enteric pathogens was encouraging, and does not support the allegation of organic foods being of high risk due to the farming methods used. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Enteric pathogens; Aeromonas spp.; Organic vegetables

1. Introduction With the increase in customer demand for freshrminimally processed, preservative-freerorganic vegetables, and the increase in importation of fresh produce from countries where hygiene standards may be compromised ŽBeuchat, 1996., there has been heightened interest in outbreaks of human

) Corresponding author. Tel.: q44-028-90-366171; fax: q44028-90-366977. E-mail address: [email protected] ŽM.A.S. McMahon..

gastroenteritis that may be associated with such produce ŽKirov, 1993; Pedroso et al., 1997.. There are a number of reports indicating that raw vegetables may harbour potential foodborne pathogens ŽNguyen-the and Carlin, 1994; Beuchat, 1996.. Listeria monocytogenes ŽSchlech et al., 1983., Salmonella ŽDoyle, 1990., and Escherichia coli ŽNguyen-the and Carlin, 1994. have been isolated from raw vegetables. Vegetables can become contaminated with such pathogenic organisms while growing, during harvest, from post-harvest handling, or during distribution. Aeromonas spp. are ubiquitous in the environment, where fresh and salt water ŽAraujo et al.,1991;

0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 5 3 5 - 9

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Amitpal et al., 1992., chlorinated drinking water ŽPicard and Goullet, 1987; Havelaar et al., 1992., and animal faeces ŽGray et al., 1990. are sources for the contamination of food products. Aeromonas species have also been isolated from a wide range of foods ŽKnochel and Jeppese, 1990; Gobat and Jemmi, 1993; Hudson et al., 1994., including vegetables ŽCallister and Agger, 1987.. Aeromonas spp. isolated from vegetables have been shown to represent a potential risk to consumers health ŽPedroso et al., 1997., due to their possible pathogenicity ŽJanda and Duffey, 1988; Cahill, 1990., and ability to grow at refrigeration temperatures ŽMcMullen and Stiles, 1992; Davies and Slade, 1995.. Monteil and HarfMonteil Ž1997. reported that Aeromonas spp. were probably one of the leading causes of bacterial enteritis during summer months, affecting mainly young children and travellers. Other authors have reported increased incidences of Aeromonas spp. in water ŽNishikawa and Kishi, 1988. and human faecal specimens ŽBurke et al., 1994. during the summer months. The correlation between summer season, increased water contamination levels and increased numbers of gastroenteritis cases may be associated, in part with the increased consumption of minimally processed or raw vegetables in salads ŽFrancis et al., 1999.. Organic vegetables have been considered by some to represent an increased risk to public health, due to the method of cultivation and processing, where natural fertilisers such as animal manure are used, and where no chemical treatments are employed to reduce the microbiological loading of the raw product, or to extend its shelf life. In addition to environmental colonisation, there may be an increased potential for such vegetables to become and remain contaminated with potentially pathogenic species from enteric sources. This study investigated the occurrence of selected enteric pathogens and aeromonads in organic vegetables in Northern Ireland, and discusses the implications of the findings.

2. Materials and methods A total of 86 organic vegetable samples from large supermarket chains that are the main retail source of organic produce and from organic farms,

located in various regions of Northern Ireland, was examined for the presence of selected enteric pathogens. Organic vegetable samples were collected over a period of 7 weeks. Where the vegetable type was purchased in a multipack form Žmore than one example of an individual vegetable type in each pack —carrot, carrot Žcut., mushroom, tomatoes, cherry tomatoes, pepper, courgette, asparagus, red onion, onion, spring onion, potato, alfalfa sprouts and water cress., a number of examples of the vegetable were randomly selected from each multipack, and 25- or 1-g samples collected as appropriate. Individual examples of vegetable types were also purchased—lettuce, cucumber, broccoli, cabbage, celery, cauliflower and turnip. All vegetables were whole unless stated. All samples were aseptically taken from the internal flesh or leaves of the vegetable where appropriate. 2.1. Isolation of E. coli O 157 A 25-g sample was removed from each vegetable, and pummelled in a stomacher with 225-ml Modified Tryptone Soya Broth ŽOxoid., the homogenate was incubated at 41.5 8C for 22 h. Dynabeads O157 ŽDynal, UK. were used according to manufacturer’s instructions to concentrate E. coli O 157 cells. A 50-ml sample was subcultured onto Cefixime-Tellurite Sorbitol MacConkey Agar ŽCT-SMAC, Oxoid. and incubated for 24 h at 37 8C. Positive colonies were transferred to Nutrient Agar ŽOxoid. and MacConkey Agar ŽOxoid. and identified by serological and biochemical tests. 2.2. Isolation of Salmonella species A 25-g sample was pummelled in a Stomachere with 225-ml Buffered Peptone Water ŽOxoid., and the homogenate was pre-enriched at 37 8C for 18 h. A 100-ml sample was subcultured into 10 ml of Rappaport Vassiliadis Broth ŽOxoid., and enriched at 41.5 8C for 22 and 48 h. One milliliter of the pre-enrichment broth was simultaneously inoculated into 10-ml Selenite Cysteine Broth ŽOxoid. and enriched at 37 8C for 22 and 48 h. Both enrichment broths were streaked onto XLD and Brilliant Green agars ŽOxoid., and incubated at 37 8C for 22 h. If present, two typical Salmonella colonies from each

M.A.S. McMahon, I.G. Wilsonr International Journal of Food Microbiology 70 (2001) 155–162

plate were subcultured into Urea Broth ŽOxoid. and MacConkey Agar, and identified by biochemical and serological reactions.

157

44 8C for 18 h. Plates were examined for blue colonies; these were confirmed as E. coli by growth at 44 8C on Tryptone Bile Agar ŽOxoid. and bglucuronidase production.

2.3. Isolation of Campylobacter species 2.5. Isolation of Listeria species A 25-g sample was pummelled in a Stomachere with 225-ml Exeter Broth ŽOxoid., and enriched by incubation at 37 8C for 24 h, followed by incubation at 42 8C for a further 22 h. The broths were streaked onto CCDA Agar ŽOxoid., and incubated microaerophilically at 37 8C for 24 h. Suspect colonies were subcultured onto 2 = Columbia Agar ŽOxoid. plates and incubated at 37 8C for 24 h, one aerobically and one anaerobically. Suspect colonies were confirmed by serological testing. 2.4. Isolation of E. coli A 1-g sample was pummelled in a Stomachere in 10-ml Peptone Saline Diluent ŽOxoid.. The homogenate was cultured onto TBX Agar ŽOxoid., and incubated at 30 8C for 4 h, followed by incubation at

A 1-g sample was pummelled in a Stomachere in 10-ml Peptone Saline Diluent ŽOxoid.. The homogenate was cultured onto Oxford Agar ŽOxoid., and incubated at 30 8C for 48 h. Five presumptive colonies were subcultured onto Blood Agar ŽOxoid. and identified using biochemical tests. 2.6. Isolation of Aeromonas species A 25-g sample was pummelled in a Stomachere with 225-ml of Tryptone Soya Broth ŽOxoid., and then incubated at 28 8C, with agitation at 150 rpm for 24 h. A-1 ml sample of homogenate was serially diluted with Ringers solution ŽOxoid.. One milliliter of each dilution was used to inoculate Starch Ampicillin Agar ŽPalumbo et al., 1985., with the plates

Table 1 Species distribution of Aeromonas isolated by source Source

No. of samples

Aeromonas spp.a

A. schubertii b

A. trotab

A. hydrophilab

A. caÕiae b

A. Õeronii bv Õeronii b

Alfalfa sprouts Asparagus Broccoli Cabbage Cauliflower Carrot Carrot Žcut. Celery Cherry tomatoes Courgette Cucumber Lettuce Mushroom Onion Pepper Potato Red onion Spring onion Turnip Water cress

1 1 4 4 1 13 2 3 4 3 6 8 12 3 6 3 1 2 3 1

1 Ž100%. 1 Ž100%. 1 Ž25%. 0 1 Ž100%. 9 Ž69%. 0 1 Ž33%. 1 Ž25%. 1 Ž100%. 4 Ž67%. 2 Ž25%. 1 Ž8.3%. 0 3 Ž50%. 0 0 0 1 Ž33%. 1 Ž100%.

– – 1 Ž100%. – 1 Ž50%. 6 Ž67%. – 1 Ž50%. 1 Ž100%. 1 Ž100%. 3 Ž60%. 1 Ž100%. 1 Ž100%. – 3 Ž67%. – – – – –

– – – – 1 Ž50%. 1 Ž11%. – 1 Ž50%. – – 1 Ž20%. – – – 1 Ž33%. – – – – –

1 Ž100%. – – – – – – – – – 1 Ž20%. – – – – – – – 1 Ž100%. 1 Ž100%.

– – – – – 2 Ž22%. – – – – – 1 Ž100%. – – – – – – – –

– 1 Ž100%. – – – – – – 1 Ž100%. – – – – – – – – – – –

a b

Number of positives. Figures in parentheses are the percentages of positive samples. Numbers of strains isolated. Figures in parentheses are relative percentages of isolates of each species.

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being incubated at 28 8C for 24 h. The Starch Ampicillin Agar plates were examined for colonies exhibiting typical Aeromonas colonial morphology. Such colonies were recorded as presumptive Aeromonas spp. Four such colonies were removed from representative plates from each positive sample and sub-cultured onto duplicate Nutrient Agar plates. A Or129 vibriostatic disc ŽOxoid. was applied to one plate. The second plate was inoculated by streaking to form an area of discrete colonies. The Nutrient Agar plates were incubated at 28 8C for 24 h. The isolates were examined for Gram, catalase, oxidase and Or129 sensitivity reactions. Suspect Aeromonas isolates were gram-negative, oxidase, catalase-positive bacilli, resistant to vibriostatic agent Or129. Such isolates were confirmed to the species level using the Aerokey 11 identification key of Carnahan et al. Ž1991.. Reference culture Aeromonas hydrophila NCIMB 9240 obtained from the National Collection of Industrial and Marine Bacteria, was used as a positive control for all biochemical tests.

3. Results The majority of the organic vegetable samples was sourced from retail facilities Ž95%. and as such was commercially packaged. The remainder Ž5%. was obtained directly from organic farms in a nonpackaged state. No Salmonella, Campylobacter, E. coli, E. coli O157 or Listeria species were isolated from the organic vegetables examined. However, Aeromonas spp. were present in 34% of the organic vegetables tested. Of the 64% of vegetables that would be regarded as requiring minimal processing

prior to consumption, e.g., washing, 41% contained Aeromonas spp. Tables 1 and 2 present the distribution of Aeromonas species in the organic vegetables tested. Of the total number of organic vegetables examined, the most commonly isolated species of Aeromonas was A. schubertii with 21.0% of all samples contaminated with this species; 5.8% of samples contained A. hydrophila, 5.8% A. trota, 3.5% A. caÕiae and 2.3% contained A. Õeronii biovar Õeronii.

4. Discussion There is a great lack of information available on the microbiological quality of foods produced under organic certification schemes. This survey was conducted as part of a national survey of over 3000 ready-to-eat organic vegetables, in which no pathogens were found ŽMitchell, personal communication.; the Aeromonas survey was an extension to this study. Listeria, E. coli O157, E. coli, Salmonella and Campylobacter were not found in the organic vegetables examined. L. monocytogenes has been isolated from many ready-to-use ŽRTU. vegetables at rates ranging from 0 ŽFenlon et al., 1996. to 44% ŽDoris and Seah, 1995.. It is present in low numbers in many foods and requires sensitive enrichment methods for detection. A previous survey in Northern Ireland showed that 2% of conventionally farmed ready-to-eat vegetable products contained Listeria spp. ŽWilson, 1995.. Listeriosis outbreaks have previously been epidemiologically associated with the consumption of raw salad vegetables ŽHo et al.,1986..

Table 2 Speciation of Aeromonas spp. isolated from organic vegetable samples Aeromonas spp.

Number of samples Aeromonas spp. isolated from

Samples examined containing each species Ž% of total.

Proportion of species isolated Ž%.

A. schubertii A. trota A. hydrophila A. caÕiae A. Õeronii biovar Õeronii

18 5 5 3 2

21.0 5.8 5.8 3.5 2.3

54.5 15.2 15.2 9.1 6.0

M.A.S. McMahon, I.G. Wilsonr International Journal of Food Microbiology 70 (2001) 155–162

Soriano et al. Ž2000. reported the isolation of E. coli from restaurant prepared lettuce. Outbreaks of food poisoning associated with E. coli O157, the principal reservoir for which is thought to be cattle faeces ŽDoyle et al., 1997., has been related to the consumption of vegetables and salads ŽDoyle et al., 1997; Wilson and Heaney, 1999.. Salmonella species have been isolated from a range of vegetables ŽGarcia-Villanova Ruiz et al., 1987. and Salmonella food poisoning outbreaks have been associated with the consumption of plant foods such as tomatoes ŽWood et al., 1991.. Due to the lack of available information on the pathogenic status of bacteria isolated from organic vegetables, direct comparisons with conventional vegetables cannot be made. In many of the previously mentioned cases, inadequate hygienic practices during processing may have been responsible for the outbreaks, as opposed to poor microbiological quality of the vegetables. However, Aeromonas spp. were isolated from 29 Ž34%. of the organic vegetables tested in this study. The literature contains numerous reports of the presence of Aeromonas species on vegetables. Of conventionally farmed vegetables, Callister and Agger Ž1987. detected A. hydrophila on almost all of the vegetables examined and suggested that retail vegetable produce may be an important source of A. hydrophila gastroenteritis. Saad et al. Ž1995. reported that 47.8% of vegetables tested contained Aeromonas spp. Aeromonads have also been isolated from restaurant prepared lettuce ŽSoriano et al., 2000., pre-made salads ŽFricker and Tompsett, 1989. and commercial vegetable salads ŽGarcia-Gimeno et al., 1996.. Due to certain practices used in producing and processing organically grown produce Že.g., manuring instead of chemical fertilisers, prohibition of chlorine dipping., the fact that microbial populations are reported to be higher in organic conversion soil plots compared to conventionally farmed soil plots ŽSilvapalan et al., 1993., and the ubiquitous nature of Aeromonas spp. in the environment, one might expect the occurrence of aeromonads in organic vegetables to be greater than that of conventional vegetables. This study suggests that this is not the case, i.e., the occurrence of aeromonads in organic vegetables is similar to, or less than that of conventional vegetables. Organic farming in the UK is the subject

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of a number of misconceptions. It is regulated by EEC Regulation No. 2092r91 and a strict program of certification is enforced in the United Kingdom by local authority Trading Standards Officers and the Register of Organic Food Standards ŽUKROFS.. EU approved certification bodies such as Soil Association Certification, which regulate the products and processes they certify. While human enteric pathogens have been transmitted to plant materials by manure, documented cases have been with conventional farming systems ŽFenlon et al., 1996; Doyle et al., 1997.. Organic certification requires manure management protocols that ensure manure is composted with a high rise in temperature for at least 3 months, which eliminates most pathogens. Organically farmed animals may have a lower pathogen load in their faeces due to the high animal welfare standards and the prohibition of antibiotic use ŽPavia et al., 1990.. Additionally, organic practices preclude adding manure directly to crops which may be eaten raw. The present study suggests that the methodology employed in organic farming may not increase aeromonad-contamination of produce, although a study enumerating Aeromonas spp. in cohorts of organic and conventional vegetables, would be necessary to verify this. Many Ž64%. of the organic vegetables examined were Aready-to-eatB after minimal processing and Aeromonas spp. were isolated from 41% of these vegetables. Conventional minimally processed vegetables are fresh, raw vegetables usually trimmed, peeled or cut, washed and disinfected using chemical additives prior to packaging ŽFrancis et al., 1999.. Unlike conventionally prepared ready-to-use vegetables, the criterion for organic produce is to provide a product where no chemical treatments have been employed either during growth or processing. Therefore, Aready-to-eatB organic vegetables are likely to retain much of their indigenous microflora even after minimal processing. This study indicates that in a large proportion of samples, organisms such as Aeromonas spp., which may potentially be pathogens, form part of the natural flora. All the Aeromonas species isolated in this study have previously been reported to be potentially pathogenic and may cause gastrointestinal infections in humans. A. hydrophila, A. Õeronii biovar Õeronii,

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A. trota and A. schubertii have all been associated with gastroenteritis ŽHanninen et al., 1995; Merino et al., 1995; Janda and Abbott, 1998.. A. caÕiae has been regarded by some authors as a non-pathogenic species ŽMillership et al., 1986; Merino et al., 1995.. However, Namdari and Bottone Ž1990. and Karanakaran and Devi Ž1994. reported that, as with other pathogenic species of Aeromonas, A. caÕiae could also be regarded as a pathogen due to the expression of virulence factors such as b haemolysin, cytotoxins and enterotoxins. Most hospital laboratories do not examine faeces for aeromonads, as they are not accepted to be of common clinical significance. It is therefore not possible to estimate the prevalence of illness due to these organisms. The proportions of individual species isolated in this study—schubertii Ž54.5%., hydrophila Ž15.2%., trota Ž15.2%., caÕiae Ž9.1%. and Õeronii biovar Õeronii Ž6.0%., vary from those of other studies. Of the Aeromonas isolates obtained from conventionally farmed vegetables by Saad et al. Ž1995., 96.5% were A. caÕiae and 2.8% were A. hydrophila. As well, 10.4% of isolates from ready-to-eat lettuce were identified as A. hydrophila ŽSoriano et al., 2000.. Nishikawa and Kishi Ž1988. isolated A. hydrophila Ž37%. and A. caÕiae Ž63%. from vegetables. A. caÕiae was amongst the majority of bacteria isolated from spinach ŽBabic et al., 1996. and Pedroso et al. Ž1997., isolated A. caÕiae and A. hydrophila from escarole, watercress and lettuce. By contrast, no Aeromonas spp. was isolated from the vegetables examined by Krovacek et al. Ž1992.. There are a number of possible explanations for the differences in the Aeromonas species profiles reported by different authors. The species identification schemes vary between authors, where some of the schemes have been superseded by more accurate systems. As with this study, the majority of clinical laboratories identify Aeromonas spp. following the Aerokey II system of Carnahan et al. Ž1991., where six species of Aeromonas would be detected. Other schemes such as that set out by Bergey’s manual 1984 ŽPopoff, 1984., used by Nishikawa and Kishi Ž1988., identify Aeromonas species into three groups—hydrophila, sobria and caÕiae. There may be a differential geographical dispersion of Aeromonas species between studies carried out in different countries. Many authors have found that the occurrence of particular

species of Aeromonas in foods varies between and within countries. In Australia, Kirov Ž1993. reported A. hydrophila and A Õeronii biotype sobria to be the predominant environmental Žfood and water. isolates, whereas in Japan, A caÕiae was found to be the predominant species ŽNishikawa and Kishi, 1988., and A. hydrophila predominated in vegetable products in the USA ŽCallister and Agger, 1987.. The findings of these workers suggest that there may be a distinct geographical distribution of particular species of Aeromonas in foods worldwide. Aeromonas species were isolated from the majority of vegetables types examined, except cut carrots, onion-type vegetables, cabbage and potato. The survey was not designed to identify differences between vegetables, and consequently the numbers of each vegetable type were too small to test the statistical significance of differences. However, if vegetables are amalgamated into groups containing six or more samples, the Fisher exact P-value of 0.007 suggests a variation in positivity between vegetables. Carrot and lettuce differed significantly from one another Ž P s 0.004., but the absence of isolates from certain vegetables is of uncertain significance. Further work will be required to establish if this effect is real. Antibacterialrinhibitory substances within these vegetables may be released during harvesting, preparation, or stomaching, and there may be components of the microflora that express antimicrobial compounds to enhance their own competitiveness. There are a number of reports that indicate the antibacterial activity of cut carrots and carrot juice ŽNguyen-the and Lund, 1991; Abdulraouf et al., 1993; Beuchat et al., 1994.. Although the nature of the antibacterial factor is unknown, antimicrobial phenolic or polyacetylenic compounds have been isolated from carrot roots ŽNguyen-the and Lund, 1991.. Such compounds have antibacterial activity towards Listeria spp. and E. coli; the effect on Aeromonas spp. is unknown. Members of the onion family Ž Allium. also possess an antimicrobial agent—allicin ŽTariq and Magee, 1989., which may be inhibitory to Aeromonas spp. Jacxsens et al. Ž1999. reported that the growth of psychrotrophic pathogens such as Aeromonas spp. on fresh-cut vegetables was influenced by the type of vegetable examined, where inhibition of growth in Aeromonas spp. occurred with brussel sprouts—members of the cabbage fam-

M.A.S. McMahon, I.G. Wilsonr International Journal of Food Microbiology 70 (2001) 155–162

ily Ž Brassica.. Other vegetables may also possess natural antimicrobial compounds that have an antimicrobial effect on Aeromonas spp., thus reducing their culturability. Many of the indigenous microflora of raw vegetables, for example, the lactic acid bacteria, may be capable of expressing bacteriocins—a heterogeneous group of proteins with antibacterial activity, Lactococcus lactis subsp. lactis cultures for example have an inhibitory effect on A. hydrophila strains ŽSantos et al., 1996.. This study was not designed to compare organic and conventionally farmed vegetables. The results from this study indicate that Aeromonas spp. is frequently detected in organic vegetables. However, the absence of accepted enteric pathogens was encouraging and does not support the suggestion of organic foods being of high risk due to the farming methods used. Acknowledgements Our sincere thanks to Northern Ireland Local Authority Environmental Health Departments for the collection of organic vegetables used in this study, and to Nick Andrews, PHLS Statistics Unit for the statistical advice. References Abdulraouf, U.M., Beuchat, L.R., Ammar, M.S., 1993. Survival of Escherichia coli O157-H7 on salad vegetables. Appl. Environ. Microbiol. 59 Ž7., 1999–2006. Amitpal, T., Ramamurthy, T., Asit, R., Sudhir, C., Yoshifumi, T., Balakrish Nair, G., 1992. Virulence traits of Aeromonas strains in relation to species and source of isolation. Zbl. Bakt. 276, 418–428. Araujo, R.M., Arribas, R.M., Pares, R., 1991. Distribution of Aeromonas species in waters with different levels of pollution. J. Appl. Bacteriol. 71, 182–186. Babic, I., Roy, S., Watada, A.E., Wergin, W.P., 1996. Changes in microbial populations in fresh spinach. Int. J. Food Microbiol. 31 Ž1., 107–119. Beuchat, L.R., 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59, 204–216. Beuchat, L.R., Brackett, R.E., Doyle, M.P., 1994. Lethality of carrot juice to Listeria monocytogenes as affected by pH, sodium chloride and temperature. J. Food Prot. 57, 470–474. Burke, V., Robinson, J., Cooper, M., Beaman, J., Partridge, K., Peterson, D., Gracey, M., 1994. Biotyping and virulence factors in clinical and environmental isolates of Aeromonas species. Appl. Environ. Microbiol. 47, 1146–1149.

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