Bacterial quality and safety of packaged fresh leafy vegetables at the retail level in Finland

International Journal of Food Microbiology 232 (2016) 73–79

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Bacterial quality and safety of packaged fresh leafy vegetables at the retail level in Finland L.-L. Nousiainen ⁎, S. Joutsen, J. Lunden, M.-L. Hänninen, M. Fredriksson-Ahomaa Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 26 November 2015 Received in revised form 12 May 2016 Accepted 14 May 2016 Available online 24 May 2016 Keywords: Leafy vegetables Packaging Bacteria Quality Safety Labelling

a b s t r a c t Consumption of packaged fresh leafy vegetables, which are convenient ready-to-eat products, has increased during the last decade. The number of foodborne outbreaks associated with these products has concurrently increased. In our study, (1) label information, (2) O2/CO2 composition, (3) bacterial quality and (4) safety of 100 fresh leafy vegetables at the retail level were studied in Finland during 2013. Bacterial quality was studied using aerobic bacteria (AB) and coliform bacteria (CB) counts, and searching for the presence of Escherichia coli, Listeria and Yersinia. The safety was studied by the presence of Salmonella, ail-positive Yersinia, stx-positive E. coli (STEC) and Listeria monocytogenes using PCR and culturing. Important label information was unavailable on several packages originating from different companies. The packaging date was missing on all packages and the date of durability on 83% of the packages. Storage temperature was declared on 62% of the packages and 73% of the packages contained information about prewashing. The batch/lot number was missing on 29% of the packages. Very low oxygen (O2) (b 1%) and elevated carbon dioxide (CO2) (2–22%) concentrations were measured in all packages labelled to contain a protective atmosphere. O2 and CO2 concentrations varied widely in the rest of the packages. AB and CB counts were high in the leafy vegetable samples varying between 6.2 and 10.6 and 4.2–8.3 log cfu/g, respectively. In most of the samples, the AB and CB counts exceeded 108 and 106 cfu/g, respectively. A positive correlation was observed between the AB and CB counts. E. coli was isolated from 15% of the samples and Yersinia from 33%. L. monocytogenes was isolated from two samples and ail-positive Y. enterocolitica in one. Using PCR, STEC was detected in seven samples, and Salmonella and ail-positive Y. enterocolitica in two samples each. The AB and CB mean values of products originating from different companies varied widely. High AB and CB counts and pathogenic bacteria were detected in ready-to-eat products not needing washing before use. Our study shows that the bacterial quality and safety of packaged fresh leafy vegetables is poor and label information on the packages is inadequate. More studies are needed concerning the impact of a protective atmosphere on bacterial growth, and the impact of washing for removing bacteria. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Packaged salads including fresh leafy vegetables (leafy greens) are widely consumed ready-to-eat food products (EFSA, 2014). They are mostly minimally processed including steps like cutting, washing, dewatering, packaging and storage. Several washing steps are used to cool and clean the leafy vegetables, mainly to eliminate field dirt and debris but also to decrease microbial contamination. Some microorganisms will be removed from the product during washing, but they can also be spread from contaminated parts to non-contaminated ones (Ailes et al., 2008). Processing leafy greens into fresh-cut products may increase the risk of bacterial contamination by breaking the external barrier of the produce (Francis et al., 2012). Using disinfectants to inactivate bacteria may be applicable at this stage (Lynch et al., 2009). Irradiation, which reduces microbial contamination without damaging ⁎ Corresponding author. E-mail address: liina-lotta.nousiainen@hel.fi (L.-L. Nousiainen).

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.05.020 0168-1605/© 2016 Elsevier B.V. All rights reserved.

the texture and colour of the produce, is currently not permitted for use on leafy vegetable in most European countries (EU, 2009). The maximum radiation dose for vegetables is 1 kGy which has shown to be effective for elimination of foodborne pathogens in leafy greens. However, the efficacy is influenced by the atmosphere in which it is packaged (Olaimat and Holley, 2012). Fresh leafy vegetables are products with a short shelf-life, especially when stored in inappropriate conditions. Storage temperature and humidity are very important factors influencing the quality and safety of leafy greens (Francis et al., 2012). Packaging effectively prevents water loss but due to respiration, vegetables consume oxygen (O2) and produce carbon dioxide (CO2), thereby modifying the gas composition within the package. Modified atmosphere packaging (MAP) techniques, including passively- and actively-altered environments, are commonly used for fresh produce (Caleb et al., 2013; Sandhya, 2010). MAP can extend the shelf life of fresh vegetables considerably compared to atmosphere packaging. Packaging is also applied to ease product handling and for hygienic purposes. However, concerns have been raised of

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possible growth of pathogens such as Salmonella, Listeria monocytogenes and stx-positive Escherichia coli (STEC) in fresh and fresh-cut produce under MA during prolonged storage especially at abusive temperatures (Caleb et al., 2013; Francis et al., 2012). The minimum labelling requirements of packaged vegetables in the EU includes clear product type markings including ingredients, country of origin, quality class, net weight, and the company name and address on each package (EU, 2011). However, storage temperature information and best before/use-by dates are also important for consumers. Furthermore, consumers need clear guidance on whether the products require washing before use. Packages should also be labelled with a batch number or with comparable information providing reliable traceability. Irradiated vegetables must be labelled. The number of reported outbreaks associated with food of non-animal origin including leafy greens has increased (EFSA, 2013). Increased consumption, large-scale production and efficient distribution during the past decade may have contributed to this increase. Outbreaks of Salmonella, STEC, enteropathogenic Yersinia (Yersinia enterocolitica and Yersinia pseudotuberculosis) and L. monocytogenes have been associated with fresh leafy greens (Callejón et al., 2015; Little and Gillespie, 2008; MacDonald et al., 2011; Marder et al. 2014; Nuorti et al., 2008; Taban and Halkman, 2011). Packaging type and storage temperature may impact the growth of these pathogens due to prolonged storage. The aim of this work was to study (1) the quality and (2) safety of packaged leafy vegetables from retail shops, (3) the label information on the packages and (4) the packaging's gas composition effect on microbial quality and safety were also studied.

Information concerning whether the product should be washed before use or whether it was ready to use was additionally checked for. 2.3. Microbiological quality Up to 100 g of each sample was transferred to a stomacher bag (Seward stomacher 400 classic bags, Sussex, UK) and 200 ml of buffered peptone water (BPW (ISO), LAB M, Kerava, Finland) was added and mixed gently by hand for 30 s. The numbers of aerobic bacteria (AB) and coliform bacteria (CB) were determined using the drop plating method (DIN, 1984). The sample was further diluted (1:10) in peptone saline water (distilled water with 0.85% NaCl and 0.1% peptone) to 10− 8. Plate count agar (PCA, Oxoid, Basingstoke, UK) and Chromocult (Merck, Darmstadt, Germany) plates for AB and coliforms, respectively, were inoculated from tenfold dilutions in duplicate. The PCA and Chromocult plates were incubated at 30 °C for 24–48 h and 37 °C for 24 h, respectively. To detect E. coli, 100 μl of the samples mixed with BPW (1:3) was plated on Chromocult and violet-red-bile (VRB, Oxoid) plates, which were incubated at 42 °C for 24 h. Furthermore, the most probable number (MPN) of E. coli was determined using the Colilert® test (IDEXX Laboratories, Westbrook, Maine) according to manufacturer's instruction, to increase the sample volume. The samples mixed with BPW were diluted tenfold and cultured in separate Colilert trays to detect the numbers of viable E. coli between 1 and 2419 in 100 ml. Coliform counts were also possible to measure using the same test. 2.4. Microbiological safety

2. Material

The presence of Salmonella, Yersinia and Listeria were studied by culturing. First, these pathogens were cultured directly on a selective agar plate: 100 μl of the sample mixed with BPW was inoculated on a xyloselysine-deoxylate (XLD, LAB M) agar for Salmonella, Oxford agar (LAB M) for Listeria and cefsulodin-irgasan-novobiocin (CIN, LAB M) agar for Yersinia isolation. Salmonella isolation continued by inoculating 100 μl of the overnight (18–20 h) enrichment (in BPW at 30 °C) on the modified semisolid Rappaport-Vassiliadis (MSRV, LAB M) agar. After 24-h incubation at 42 °C, any spreading growth (if present) found on the MSRV plate was further plated on an XLD plate, which was incubated for 24 h at 37 °C. Listeria was isolated by inoculating 100 μl of the overnight enrichment into 10 ml of Fraser broth (LAB M) and after 48-h incubation, 10 μl was inoculated on an Oxford agar (LAB M) plate which was incubated at 37 °C for 24–48 h. Yersinia was isolated on a CIN agar plate after cold enrichment for 8 d at 4 °C in peptone-mannitol-bile salt (PMB) broth (10 ml of BPW + 90 ml of PMB) and an alkaline treatment (0.5% KOH) of PMB broth for 20 s. CIN plates were incubated at 30 °C for 20–24 h. Typical colonies on XLD and CIN plates were identified using API 20E strips (bioMérieux, Marcy l'Etoile, France), and typical colonies on Oxford plates using API Listeria strips (bioMérieux). The presence of Salmonella enterica, STEC and enteropathogenic Yersinia spp. (ail-positive Y. enterocolitica and Y. pseudotuberculosis) were examined using real-time PCR. DNA was extracted from the overnight enrichment of BPW using Chelex®100 resin (Bio-Rad, Hercules, California). Shortly, 100 μl of the overnight enrichment was centrifuged at full speed (13,000 × rpm) for 1 min. The supernatant was removed

2.1. Sampling and gas composition One hundred packaged fresh leafy green vegetables were bought from retail stores in Finland in 2013 (Table 1). All products were packaged in a bag. The dominant product type was mixed leafy greens (60%) containing frequently arugula, radicchio, mizuna, romaine and spinach. The rest of the products were spinach (19%) and different leafy greens (21%: head lettuce, frisee, arugula, kale and romaine). Most (57/60; 95%) of the products sampled between January and April were imported, while domestic products were mainly (33/40; 83%) sampled in August and September due to low domestic production during the winter season. The imported products mainly originated from Italy and Spain. A maximum of five different packages were tested per week. All packages were from different batches. The gas composition was measured using a gas sensor (Checkpoint, PBI Dansensor, Ringstedt, Denmark) before microbiological studies (Table 1). 2.2. Labelling Product type describing the product, product ingredients, country of origin, quality class of the vegetable(s), net weight, batch/lot number, company name and address, nutritional information, packaging type (protective atmosphere), packaging date, date of durability/best before date and storage temperature of the package were recorded (Table 2).

Table 1 Origin of packaged fresh leafy vegetables from retail level studied in Finland year 2013. Number of packages (%) sampled

Number of packages (%) with

Origin of packages

2013

January–April

August–November

O2: b1% CO2: 2–22%

O2: 1–18% CO2: 2–10%

O2: N18% CO2: b2%

Domestic Imported All

36 64a 100

3 57 60

33 7 40

11 8 19

15 24 39

10 32 42

a

(8.3) (89.1) (60.0)

Italy:39, Spain:15, Sweden:8, France:1, Netherlands:1.

(91.7) (10.9) (40.0)

(30.6) (12.5) (19.0)

(41.7) (37.5) (39.9)

(27.8) (50.0) (42.0)

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Table 2 Label information found on the fresh leafy vegetable packages at retail level. Number of Information

Packages (n = 100)

Product types (n = 52)

Companies (n = 22)

Company namea Company addressa Product typea Country of origina Net weighta Product ingredientsa Batch/lot numbera Storage temperature Quality classa Wash before use Nutrition information Date of durability (best before) Ready to use Packed in a protective atmosphere Date of packaging

100 100 100 98 98 97 71 62 62 58 29 17 15 13 0

52 52 52 50 50 50 35 35 27 23 11 12 10 9 0

22 22 22 20 22 22 11 15 12 9 6 4 3 2 0

a

The minimum labelling requirements of packaged vegetables in the EU.

and the pellet was re-suspended in 100 μl of a 5% suspension of deionized water and Chelex®100 according to the manufacturer. The suspension was incubated at 56 °C for 20 min and then at 99 °C for 10 min. After 3-min centrifugation at full speed, approximately 50 μl of the supernatant was transferred to a new tube and stored at − 20 °C. For realtime PCR screening, the ttr of Salmonella spp. (Malorny et al., 2004), ail of Y. enterocolitica and Y. pseudotuberculosis (Thisted Lambertz et al., 2008a,b) and stx1 and stx2 of STEC (Sharma and Dean-Nyström, 2003) were amplified. The total reaction volume for PCR was 25 μl containing 1× of a ready-to-use mix (iQ™SYBRGreen Supermix, Bio-Rad), 200 nM of primers (Oligomer, Helsinki, Finland) and 2 μl of template. A threestep protocol (denaturation at 95 °C for 10 s, annealing at 58 °C for 10 s and elongation at 72 °C for 30 s) with 40 cycles followed by a melting curve analysis was used. The fluorescence intensity of the SYBRGreen was studied using the CFX96™ Real-Time PCR Detection System (Bio-Rad). The sample was considered positive when the threshold cycle (Ct) was below 38 and a specific melting temperature (Tm) was observed.

2.5. Statistical analyses Statistical analyses were performed using SPSS 23 (IBM SPSS Software). All microbial counts were log transformed for statistical analyses. The Mann-Whitney U test was used to analyse the significance of the difference between AB and CB mean values of products with low and high O2 concentrations. The correlation between the AB and CB counts was analysed using Spearman's rho. Non-parametric tests were used because the AB and CB counts were not normally distributed (Kolmogorov-Smirnov test of normality p b 0.05). Fisher's exact test was used to analyse the relation between the high and low CB counts and the presence of E. coli, Yersinia and Listeria. A confidence level of 95% was applied. Half of the detection limit was used as the estimated count if the bacterial count was below the detection limit.

3. Results The label information on 100 packaged fresh leafy vegetables from 22 companies was recorded (Table 2). Name and contact information of the company, product type, country of origin, net weight and vegetable ingredients were included on all or nearly all packages. However, the packaging date was missing from all packages and the durability date was found on only 17% of the packages originating from 4 (18%) companies. Batch/lot number, quality class and storage temperature were also missing from several products (Table 2). A recommended storage temperature below 6 °C was mentioned on only 21% of the packages. Two companies (9%) reported the use of a protective atmosphere. Fifteen products from three (14%) companies were ready to use without need for further washing according to the labelling. Information about the need for washing the produce before use was missing from 27% of the packages. The O2 and CO2 concentrations in the packages varied from b0.1 to 20.7% and b0.1 to 22.2%, respectively (Table 3). The O2 concentrations of 19 packages were very low (b 1%) while the CO2 concentrations were elevated, and 13 of these packages were packed with a protective atmosphere according to the label information provided. The O2 concentrations of 42 packages were high (over 18%) while CO2 concentrations were low (below 2%). The mean AB and CB values of the 100 packaged fresh leafy greens were 8.6 (± 1.07) log cfu/g and 6.3 (± 1.18) log cfu/g, respectively. The median was slightly higher: 8.9 log cfu/g for AB and 6.5 log cfu/g for CB. The bacterial counts varied substantially from between 6.2 and 10.6 log cfu/g for AB and between 4.2 and 8.3 log cfu/g for CB (Table 3). The CB mean value of 7.0 log cfu/g in packages with very low O2 concentrations was significantly (Mann-Whitney U test; p = 0.006) higher than in packages with over 1% of O2 (Table 3). A positive correlation (Pearson correlation; r = 0.661, p b 0.001) was observed between the AB and CB counts. Most (74%) packaged fresh leafy vegetables having more than 108 AB/g were mixed leafy greens containing arugula (34/74), spinach (25/74),

Table 3 Mean values of aerobic and coliform bacteria in 100 packaged fresh leafy vegetables at retail level. Mean values (log cfu/g) O2/CO2 concentrations in the package

Bacteria

All samples (n = 100)

Aerobic Coliform

8.6 6.3

a

Minimum and maximum counts.

(6.2–10.6)a (4.2–8.3)

O2: b1% CO2: 2–22% (n = 19)

O2: 1–18% CO2: 2–10% (n = 39)

O2: N18% CO2: b2% (n = 42)

8.9 7.0

8.4 6.2

8.7 6.2

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Table 4 Counts of aerobic and coliform bacteria in 100 packaged fresh leafy vegetables. Number of samples with bacterial counts (log cfu/g) between Bacteria

4.2–5.0

5.1–6.0

6.1–7.0

7.1–8.0

8.1–9.0

9.1–10.0

10.1–10.6

Aerobic Coliform

NSa 16

0 20

11 30

15 29

35 5

35 NS

4 NS

a

NS = not studied.

radicchio (18/74), mizuna (12/74), lollo rosso (10/74) and romaine lettuce (10/74). The number of CB exceeded 107 cfu/g in 34% of the packages (Table 4). In total, 64% of the packaged fresh leafy greens had more than 106 CB/g. E. coli was found in 15% of the leafy vegetable samples using the Colilert® test based on the MPN procedure (Table 5). No E. coli was found on the Chromocult or VRB plates. The MPN of E. coli in 100 ml was low varying between 3 and 6533 and the median was 34 MPN/ 100 ml. Yersinia spp. were frequently isolated from the leafy greens: 33% of the packages were contaminated (Table 5). Y. enterocolitica and Yersinia frederiksenii were the most common species identified in 13 (40%) and 15 (45%) of the Yersinia-positive samples, respectively. All Y. enterocolitica isolates belonged to biotype 1 A not associated with human disease; however, one isolate carried the ail gene which is typically found only in biotypes 1B and 2–5 associated with disease. Listeria spp. was isolated from 4% of the products and two of these were contaminated with L. monocytogenes serotype 1/2a. The number of both Yersinia spp. and Listeria spp. was under 100 cfu/g and found only after enrichment. Using real-time PCR, STEC was detected in seven samples while stx2 was found in five and stx1 in two samples (Table 5). Furthermore, Salmonella and ail-positive Y. enterocolitica were detected in two samples each using PCR. E. coli was isolated significantly (Fisher's exact test, p = 0.03) more frequently in packages with CB counts exceeding 107 cfu/g (Table 6). Yersinia spp. was also isolated more often from packages with high CB counts (N 106 cfu/g), but the association was not significant. Both packages contaminated with L. monocytogenes contained a high number of CB. In total, 35% of the packages originating from 13 (59%) companies were contaminated with Yersinia or/and Listeria (Tables 7). The mean values of AB varied between 7.1 and 9.5 log cfu/g and CB between 4.5 and 7.5 log cfu/g in packages from companies with contaminated packages (Table 7). The mean values were slightly lower in packages from companies with no contaminated packages: AB mean values varying from 6.2 to 9.2 log cfu/g and CB from 4.2 to 6.3 log cfu/g. E. coli was isolated from packages originating from companies with and without contaminated packages: it was isolated from the packages of four (4/13, 31%) companies with contaminated packages and from three (3/9, 33%) companies with not contaminated packages. However, E. coli

Table 5 Occurrence of indicator and pathogenic bacteria in 100 fresh leafy vegetable samples detected by culturing and PCR. Bacterium

Indicator bacteria Escherichia coli Yersinia spp. Listeria spp. Pathogenic bacteria Salmonella spp. ail-positive Yersinia enterocolitica stx-positive Escherichia coli Listeria monocytogenes a

NS = not studied.

Number of samples positive by Culturing

PCR

15 33 4

NSa NS NS

0 1 0 2

2 2 7 NS

was frequently isolated in the packages of two companies (I and J). The mean values of AB and CB were also high in the packages of these two companies. Both companies recommend washing before use but company I did not include storage temperature instructions on the label. Three companies (B, E and O) produce ready-to-eat products not needing washing before use.

4. Discussion The label information of the 100 fresh leafy vegetable packages were recorded. Most of the packages met the minimum requirements in the EU regulation. Surprisingly, the batch or lot number was missing from 29% of the packages making their traceability impossible, and the quality class was missing from 38% of the packages. The storage temperature was missing from 38% of the packages, which may lead to storage at abusive temperatures thus influencing the bacterial quality and safety of the products. Inconsistent and abusive temperatures have been shown to increase the microbial growth and metabolic activities of fresh produce, and a strict cold chain along the entire distribution is thus recommended (Caleb et al., 2013; Francis et al., 2012). Storage temperature is one of the most important factors also affecting pathogen growth, and especially temperatures under 6 °C have been shown to inhibit the growth of most pathogenic species but not Listeria and Yersinia. A storage temperature of under 6 °C was recommended in only 21% of the products investigated in our study. No information regarding washing or not washing the product before use was recorded on 27% of the packages. Washing the produce in tap water removes microbes including pathogenic bacteria from exposed unbroken plant surfaces but not from damaged vegetable tissues (Francis et al., 2012). The packaging date was missing from all packages and only 17% of the packages had a durability date marked on them. Fresh vegetables have a short shelf life, but when packaged under a modified atmosphere the shelf life can be prolonged by up to 10 days (Arvanitoyannis et al., 2011; Oliveira et al., 2010a; Losio et al., 2015). Leafy vegetables have the highest bacterial counts of vegetable products without organoleptic alteration (Cardamone et al., 2015). High bacterial counts may be due to their broad surface area, which easily becomes contaminated and their rough surface, which accumulates and adheres dirt and bacteria. Several bacterial indicators including AB and CB have been used to assess the quality of fresh produce. The AB and CB counts of fresh leafy vegetables varied greatly in our study. However, there was a clear positive correlation between the AB and CB counts, showing that it is not necessary to use both indicators to assess processing hygiene or to determine the microbial quality of the leafy greens. The mean AB value (8.6 log cfu/g) was very high varying from 6.2 to 10.6 log cfu/g. Aerobic microbial counts of leafy vegetables have been extensively studied and typically they vary between 3 and 8 log cfu/g (Ailes et al., 2008; Korir et al., 2016; Oliveira et al., 2010b; Soriano et al., 2000; Wood et al., 2015). However, packaged leafy vegetables from retail shops have only occasionally been studied. AB exceeded a level of 108 cfu/g in 74% of the packaged leafy vegetables in our study. Only 4% of the ready-to-eat lettuce in Switzerland exceeded 108 cfu/g (Althaus et al., 2012). One reason for the clearly lower AB counts in Switzerland may be that the samples were taken from the production plant level and only from one company. Abusive storage temperature

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Table 6 Occurrence of Escherichia coli, Yersinia and Listeria in fresh leafy vegetable samples with different coliform bacteria counts. Counts of coliform bacteria (log cfu/g) Bacteria

Detected in 100 g (n = 100)

4.2–5.0 (n = 16)

5.1–6.0 (n = 20)

6.1–7.0 (n = 30)

7.1–8.0 (n = 29)

8.1–9.0 (n = 5)

Escherichia coli Yersinia spp. Listeria spp.

15 33 4

0 1 0

2 7 0

2 7 1

10 9 2

1 2 1

and prolonged storage time may have affected the AB counts in our study. Ailes et al. (2008) demonstrated higher microbial concentrations in packaged produce compared to those studied immediately postharvest. The mean CB value (6.3 log cfu/g) was also high in our study varying between 4.2 and 8.3 log cfu/g, while 64% of the packaged fresh leafy greens had more than 106 CB/g. CB counts in previous studies have usually been below 106 cfu/g (Korir et al., 2016; Sagoo et al., 2003; Sant'Ana et al., 2011; Soriano et al., 2000; Wood et al., 2015). In Brazil, 512 readyto-eat vegetables including lettuce from retail shops were studied and the CB counts exceeded 106 cfu/g in only 2% of the samples (Sant'Ana et al., 2011). However, all vegetables had been processed through a sanitization step during handling. Washing and rinsing fresh vegetables during processing, especially if the water is reused, have been shown to increase CB in the USA (Xu et al., 2015). Potable water is typically used to wash and rinse leafy greens in Finland. Jensen et al. (2015) have demonstrated that plain water-washing can easily spread bacteria from contaminated leaves to previously uncontaminated ones. E. coli has been used as an indicator for the faecal contamination of fresh produce (Wood et al., 2015). In our study, E. coli was detected in 15% of the samples, but the counts were low and found only by the Colilert® test, which remarkably improves the possibility of detecting E. coli due to the high sample volume. The microbial quality of all studied leafy vegetables was acceptable based on the E. coli criteria for

ready-to-eat fresh produce in the EU (n = 5, c = 2, m = 100 cfu/g, M = 1000 cfu/g) (EU, 2005). The prevalence of E. coli in leafy greens has been reported to be below 5% in several earlier studies (Ailes et al., 2008; Cardamone et al., 2015; Johannessen et al., 2002; Sagoo et al., 2003), and only some studies have found levels around 20% (Oliveira et al., 2010b; Soriano et al., 2000). However, Kahlil et al. (2015) recovered E. coli levels exceeding 100 cfu/g from all leafy green types studied in Egypt, indicating massive faecal contamination probably due to contaminated irrigation water. Contamination can also easily occur through contaminated hands, equipment and rinse water during harvesting and processing. Lehto et al. (2011) demonstrated that improved cleaning and hygiene practices in vegetable production are needed. Fresh produce can modify the atmosphere in their packages as a result of O2 consumption and CO2 production (Oliveira et al., 2010a). The O2 and CO2 concentrations in our study varied widely in the packages and did not correlate with microbial quality and safety. Very low O2 concentrations (b 1%) combined with CO2 concentrations varying between 2% and 22% were measured in 19% of the packages including packages with a protective atmosphere. It has been reported that the greatest shelf-life extension occurs at the lowest possible oxygen concentration before anaerobic respiration is initiated (Sandhya, 2010). However, the AB and CB counts in our study were high in most of the packages with very low O2 concentrations. Furthermore, the mean CB value was

Table 7 Packaged fresh leafy vegetables contaminated with Yersinia and Listeria originating from different companies. Mean values of (log cfu/g)

No. of contaminated packages Company (n = 22)

No. of packages (n = 100)

All

Ya

Lb

RTEc

Storage b6 °C

ABe

CBf

ECg-positive packages

A B C D E F I J K N O P Y 13 in total G H L M R S T U V 9 in total

4 9 20 6 8 2 6 5 4 3 4 2 1 74 9 5 5 2 1 1 1 1 1 26

3 5 4 4 4 1 3 5 1 1 2 1 1 35 0 0 0 0 0 0 1 0 0 0

3 4 4 4 4 1 3 4 1 1 2 1 1 33 0 0 0 0 0 0 0 0 0 0

0 1 0 1 0 0 1 1 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0

No Yes No NId Yes No No No NI NI Yes NI NI

Yes Yes No No Yes Yes No Yes Yes Yes Yes No Yes

No NI No NI NI NI NI NI No

Yes Yes Yes Yes No No Yes Yes No

8.9 9.3 9.3 9.0 9.0 8.9 9.5 9.3 7.7 8.7 7.7 7.1 8.8 9.0 7.8 7.0 8.7 6.3 9.2 7.7 8.6 6.2 8.1 7.7

6.9 7.5 6.7 6.3 6.5 5.0 7.4 7.2 6.4 5.0 6.4 4.5 7.0 6.6 5.4 5.0 6.3 6.1 4.2 5.8 6.2 5.3 4.2 5.5

0 2 0 0 0 0 4 4 1 0 0 0 0 11 1 0 2 0 0 1 0 0 0 4

a b c d e f g

Y, Yersinia spp. L, Listeria spp. RTE, ready-to-eat product not needing washing before use. NI, No information on washing. AB, aerobic bacteria. CB, coliform bacteria. EC, Escherichia coli.

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significantly higher (p b 0.01) in the packages with very low O2 concentrations than in packages with O2 concentration exceeding 1%. Coliforms can grow well in an anaerobic atmosphere, especially at abusive temperatures (N6°) during prolonged storage. The correct combination of packaging material, produce weight and gas composition in the package should be determined for each product type to maintain microbial quality and safety (Gil et al., 2015). However, packaging cannot correct the poor quality of vegetables, un-sanitary handling or temperature abuse. Fresh leafy vegetables in particular have been associated with foodborne outbreaks in recent years (Wood et al., 2015). However, very seldom have pathogenic bacteria been isolated from the leafy greens (Althaus et al., 2012; de Oliveira et al., 2011; Losio et al., 2015; Soriano et al., 2000; Xu et al., 2015). The pathogens of major concern in fresh produce are Salmonella spp., pathogenic E. coli and L. monocytogenes (Cardamone et al., 2015; Ceuppens et al., 2014; EFSA, 2013; Francis et al., 2012; Jensen et al., 2015; Johannessen et al., 2002; Korir et al., 2016; Little and Gillespie, 2008). In our study, Yersinia (33%) and Listeria (4%) were common findings in the packaged leafy greens; however, ail-positive Y. enterocolitica was isolated only from one mixed leafy green product containing arugula and spinach, and L. monocytogenes from two mixed leafy green products containing frisee, radicchio and/or mizuna. No Salmonella and STEC was isolated in this study; however, using PCR, Salmonella was detected in two packages and STEC in seven. These pathogens have been shown to survive under MAP conditions (Francis et al., 2012; Oliveira et al., 2010a; Taban and Halkman, 2011). Oliveira et al. (2010a) demonstrated that Salmonella and E. coli can grow on leafy greens at abusive temperatures when stored in MAP. As psychotrophic bacteria, Yersinia and Listeria can even grow in temperatures below 6 °C (MacDonald et al., 2011; Oliveira et al., 2010a). E. coli has been proven a suitable indicator for the presence of pathogens (Ceuppens et al., 2014). However, the public health significance of the presence of E. coli on produce and the influence of MAP on the growth of pathogenic bacteria remain unclear. Thirty-five percent of the packaged fresh leafy vegetables originating from 13 (59%) companies were contaminated with Yersinia and/or Listeria. The mean AB and CB values were slightly higher in packages from companies with contaminated packages which could indicate some lack of hygiene and cleaning practices during the process. Poor hygiene during production could be one reason for the high prevalence of Yersinia spp. in the packages. Interestingly, E. coli was frequently isolated in the products of two companies (I and J). The products of both companies were also contaminated with Yersinia and Listeria, and the mean values of AB (N9 log cfu/g) and CB (N7 log cfu/g) were very high. Three companies (B, E and O) produce ready-to-eat products not needing washing before use, but over 50% of these packages (11/21) were contaminated with Yersinia or Listeria which shows that washing is not a reliable approach to reduce these species. More research is needed to clarify the true impact of washing for removing bacteria during processing and before eating. 5. Conclusions Labelling was inadequate on most of the leafy vegetable packages studied in Finland; important information such as batch/lot number, storage temperature, date of packaging/durability and quality class were missing. O2 and CO2 concentrations varied widely in the packages and did not correlate with the microbial quality and safety. Both AB and CB counts were high, especially in ready-to-eat products with a protective atmosphere. A positive correlation was observed between the AB and CB counts. The number of E. coli was low, and was detected only by the Colilert® test based on the MPN procedure and not by the Chromocult or VRB plates. Several packages were contaminated with Yersinia and Listeria, including ready-to-eat products not needing washing before use according to the labelling. The results obtained in our study show that more information is needed concerning the impact of

a protective atmosphere on bacterial growth, and the impact of washing for decreasing bacterial contamination during processing.

Acknowledgements Our study was supported by the Walter Ehrström Foundation. Maria Stark and Urszula Hirvi are gratefully acknowledged for their technical assistance.

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