- Email: [email protected]
Microbiological contamination of reusable plastic bags for food transportation
Food Control 99 (2019) 158–163
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Microbiological contamination of reusable plastic bags for food transportation
T
J. Barbosaa, H. Albanoa, C.P. Silvaa,b, P. Teixeiraa,∗ a Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital 172, 4200-374, Porto, Portugal b Colégio Internato dos Carvalhos, Rua do Padrão, 83, Carvalhos, 4415-284, Pedroso, Portugal
A R T I C LE I N FO
A B S T R A C T
Keywords: Domestic environment Foodborne pathogens Reusable plastic bag Cross-contamination Antibiotic resistance
Nowadays, with so many concerns for the environment, the use of reusable plastic bags is becoming routine, instead of the use of polluting single-use plastic bags. However, this is controversial in terms of food safety, since consumers transport many different foods, which could contaminate their bags and pose a risk to their health due to cross-contamination. This study aimed to detect or enumerate several indicators/pathogens from 30 used reusable plastic (polypropylene) bags and, to evaluate their antibiotic resistance profiles after identification by 16s rRNA of each isolated microorganism. Several genera of Enterobacteriaceae, coagulase-negative staphylococci and also Listeria monocytogenes were found in the reusable plastic bags analyzed. In general, high percentages of antibiotics resistance were found, highlighting the elevated occurrence of multi-resistant isolates of coagulase-negative staphylococci and Enterobacteriaceae. This study demonstrates the level and variety of microbial contamination of some used reusable plastic bags. No correlation was found between microbial levels and the visual appearance of each bag demonstrating that appearance is not a reliable datum about the bag contamination. We believe that this study could help the competent authorities taking measures to alert consumers to good food safety practices, not only in their kitchens, but also in the bags that carry their food.
1. Introduction Reusable plastic bags for transport of groceries from the store to the consumer's home have become popular in recent years (Williams, Gerba, Maxwell, & Si, 2011). In Portugal, this trend became more noticeable from February 2015, since plastic bags began to be taxed (Martinho, Balaia, & Pires, 2017). This additional cost resulted in an increase in the use of ‘bags for life’, i.e., resistant reusable plastic bags. However, “reusable” does not mean “clean”. Reusing plastic bags is beneficial to the environment, but as already stated “the public should be mindful of the ability of bacteria to contaminate and survive for long periods of time. Bacteria can easily transfer from different types of reusable bags to the hand and back again” (Hilton, 2015). In the U.S., reusable grocery bags are considered a new cross-contamination vehicle that has the potential to pose a significant risk of bacterial cross-contamination (Byrd-Bredbenner, Berning, Martin-Biggers, & Quick, 2013). Another important issue is that using the same bag for different purposes increases the risk of contaminating the bag with a whole host of bacteria. Understanding the importance of using different bags for
∗
different purposes is an important topic for consumers. However, it was reported that one in three consumers used these bags for more than just groceries, such as gym bags, toy bags, among other uses and that 75% of consumers use these same bags for carrying raw meat and other foods (Williams et al., 2011). In addition, the same authors stated that reusable bags, if not properly washed, could play a role in the cross-contamination of foods. Contaminated reusable grocery bags could pose a foodborne illness risk - an outbreak of norovirus in a girls' soccer team was traced to a contaminated reusable grocery bag (Repp & Keene, 2012). Considering that there are few studies regarding the microbial contamination of these plastic bags, the objective of this study was to evaluate the potential for reusable bags being contaminated. For that, several indicators/pathogens were detected or enumerated, subjected to 16S rRNA identification and their antibiotic resistance profiles assessed.
Corresponding author. E-mail address: [email protected] (P. Teixeira).
https://doi.org/10.1016/j.foodcont.2018.12.041 Received 25 October 2018; Received in revised form 27 December 2018; Accepted 28 December 2018 Available online 31 December 2018 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Food Control 99 (2019) 158–163
J. Barbosa et al.
Table 1 Enumeration (log CFU/bag) and detection (presence or absence/bag) data of several microorganisms for 30 used reusable plastic bags studied. Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Area (cm2)
4838 4672 5424 4736 4928 4440 4636 4988 5031 3298 6078 5891 6078 6120 5990 4890 4890 4890 4890 4890 5301 5226 4538 4950 4928 4992 4736 5159 5226 4890
Enumeration (log CFU/bag)
Detection (presence or absence/bag)
Total microorganisms at 30 °C
Enterobacteriaceae
Enterococci
Coliforms 30 °C
E. coli
Staphylococcus coagulase -
Staphylococcus coagulase +
Listeria spp.
Listeria monocytogenes
4.8 3.4 6.3 3.5 3.9 3.5 3.6 3.9 4.3 2.8 2.5 3.4 5.9 3.2 3.2 7.3 2.4 3.7 2.4 3.5 2.4 1.9 4.9 3.0 2.4 2.0 2.5 2.2 2.5 3.9
< 1.0 3.4 4.7 < 1.0 < 1.0 3.9 2.9 < 1.0 3.9 < 1.0 < 1.0 < 1.0 5.2 3.5 3.0 4.5 < 1.0 2.8 < 1.0 < 1.0 2.8 < 1.0 < 1.0 3.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 2.7
2.7 2.0 2.8 2.0 2.5 5.7 2.9 < 2.0 < 2.0 < 2.0 < 2.0 2.0 5.1 < 2.0 2.3 5.2 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 3.7 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0
– – + – – + – – + – – – – + – + – – – – – – + – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
+ + + + + + + – + – + + + + + + + + – – + + + + + + + – + +
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– + – – – – – – – – – – – – – – – – – + – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – + – – – – – – – – – –
Legend: Presence (+) or absence (−)/bag.
2. Materials and methods
Enterobacteriaceae on violet red bile dextrose agar (VRBD, Merck) (ISO 21528-1: 2000) incubated at 37 °C for 48 h. Detection of Listeria spp. was performed in half Fraser broth (Merck), incubated at 30 °C for 24 h, followed by all confirmatory tests (ISO 11290-1: 1996). Detection of coagulase-positive Staphylococcus was performed according to a Portuguese Standard (NP 2260: 1986) in which 1 ml of each sample in BPW solution (Sampling section) was sown in simple Chapman broth (tryptone 5 gl-1; meat extract 6 gl-1; protease peptone 5 gl-1; NaCl 75 gl-1; lactose 7.5 gl-1; agar 0.5 gl-1) and incubated at 37 °C during 24 and 48 h. Cultures were then transferred to BairdParker Agar with egg yolk tellurite (BPA, Biokar Diagnostics) and plates incubated for 24–48 h at 37 °C; characteristic colonies were confirmed by coagulase test with rabbit plasma (bioMérieux, Marcy l’Etoile, France). Coliforms at 30 °C and Escherichia coli were detected according to NP 2164: 1983 and NP 2308: 1986, respectively. After incubation in simple lactose broth (Lab M) at 30 °C during 48 h, coliforms at 30 °C were detected by growth and gas production in brilliant green broth (Oxoid, Basingstoke, United Kingdom) incubated at 30 °C for 48 h, and E. coli was detected also by growth and gas production in brilliant green broth and by indole production on peptone water, both incubated at 44.5 °C for 48 h. Enumeration of total microorganisms at 30 °C, Enterobacteriaceae and enterococci were also performed for two unused plastic bags immediately after being purchased (control bags).
2.1. Sampling This study was carried out in the Metropolitan Area of Porto, Portugal, from October to December, 2015. Thirty used and two unused (control) reusable plastic bags were sampled. These bags were constituted by 100% polypropylene (type of reusable bags sold in most supermarkets), varying only in size (information about sampling area is presented in Table 1). Each of the 30 used bags belonged to a different consumer. All consumers reported that they had no criterion in the type of food transported, i.e., the analyzed bags transported all types of food, raw or processed. However, with the exception of some vegetables, all raw food (meat and fish) was inside individual packages (plastic bags or polystyrene trays with a plastic film cover). Before sampling all the bags were visually inspected by the same laboratory technician and classified as “very little use” or “extended use” and “apparently clean” or “dirty”. Samples were collected using one cotton swab moistened with sterile quarter strength Ringer's solution (Lab M, Bury, United Kingdom), which was scrubbed over all the interior area and re-suspended in 10 ml of Buffered Peptone Water (BPW, Merck, Darmstadt, Germany). All samples were transported to the laboratory in a refrigerated box and analyzed as soon as they arrived (within 24 h). 2.2. Microbiological analyses
2.3. Origin of isolates
Appropriate decimal dilutions were prepared in sterile Ringer's solution for microbial enumeration according to ISO Standards: total viable aerobic microorganisms at 30 °C on plate count agar (PCA, Pronadisa, Madrid, Spain) (ISO 4833-1: 2013) and enterococci on bile esculin azide agar (BEAA, Biokar Diagnostics, Beauvais, France) (Ferreira et al., 2006), both incubated at 30 °C for 72 h and
Colonies (about 10%) of each selective culture media were randomly selected from plates having between 15 and 150 colonies (Tabasco, Paarup, Janer, Peláez, & Requena, 2007).
159
Food Control 99 (2019) 158–163
J. Barbosa et al.
microorganisms: sample 3 with 6.3 log CFU/bag and sample 16 with 7.3 log CFU/bag. For most of the bags, counts of Enterobacteriaceae were below the detection limit of the enumeration technique. Nevertheless, counts between 3.0 and 5.0 log CFU/bag were found in a small number of samples. Also enterococci were detected on 26 samples at levels close to or below the detection limit of the enumeration technique (2.0 log CFU/bag) and only 4 samples had higher numbers (samples 6, 13 and 16 with values of ∼5.0 log CFU/bag and sample 23 with 3.0 log CFU/bag). It is important to highlight that all the inner surface of each bag was sampled, even areas which probably came into contact with food infrequently, in particular the upper areas in the larger bags. So, although results are reported per bag, these contaminants are probably concentrated in zones such as the bottom of the bags where the probability of cross-contamination to foods is higher. Each bag was classified taking into account its visual appearance at the time of sample collection (data not shown). No significant relation (P > 0.05) was found between the microbial load and the visual appearance of each bag, i.e. with “very little use” or “extended use” and “apparently clean” or “dirty”. No Staphylococcus coagulase positive were detected in any plastic bags. However, Staphylococcus coagulase negative were detected in 25 of the 30 bags analyzed. Although none of the samples revealed the presence of Escherichia coli, coliforms were detected in five bags (3, 9, 14, 16 and 23). Listeria spp. was detected in two bags and in one of them Listeria monocytogenes was present. Our results are in accordance with some of the few studies on the subject. Williams et al. (2011) found high numbers of bacteria (including fecal coliforms) in every reusable bag collected from consumers outside a grocery store. Summerbell (2009) tested 49 “used” reusable shopping bags and found that the majority had some bacterial counts, 30% elevated bacterial counts and 12% unacceptable coliform counts. Interesting to note that Summerbell (2009) and Williams et al. (2011) showed that no bacteria were found in single use plastic carryout bags or new reusable bags. Bags containing coliform bacteria could indicate that they were contaminated by raw meats or other uncooked food products; their presence demonstrates that bags become contaminated and that foodborne pathogens could exist on the bags (van Leeuwen, 2013). In fact, the major problem associated with the presence of foodborne pathogens in reusable bags is the increased risk of bacterial cross-contamination. Already in 1997, Bradford, Humphrey, & LappinScott investigated the ability of two strains of Salmonella Enteritidis PT4 to cross-contaminate from inoculated egg droplets on surfaces onto melon or beef. The authors found that cross-contamination in each portion of food occurred in 1 s, when placed on the surfaces where the egg drops were wet, and up to 1 min when the egg droplets were dry (Bradford, Humphrey, & Lappin-Scott, 1997). Also Buchholz, Davidson, Marks, Todd, and Ryser (2012) demonstrated the occurrence of crosscontamination of E. coli O157:H7, even in very low levels, between fresh-cut leafy greens. The authors proved that E. coli O157:H7 from one contaminated batch of leafy greens could easily be spread to subsequent batches of uncontaminated product in a processing facility (Buchholz et al., 2012). Despite the steel stainless be of a different material from the reusable bags, the study of Kusumaningrum, Riboldi, Hazeleger, and Beumer (2003) is no less important. The authors studied the transference of Salmonella Enteritidis, Staphylococcus aureus and Campylobacter jejuni, at different initial levels, from kitchen sponges to stainless steel surfaces and from these to slices of cucumber and roasted chicken fillet. Cross-contamination of all microorganisms occurred from surfaces to food, with or without pressure applied, with transference rates varying from 50% to more than 100% for transmission to cucumber slices, and from 25% to 100% for transmission to roasted chicken fillet slices (Kusumaningrum et al., 2003). Microorganisms obtained from the enumeration and/or detection (Table 1) were isolated and identified by 16s rRNA sequence
2.4. LAB identification by 16S rRNA sequencing Deoxyribonucleic acid (DNA) of the isolates was extracted according to the protocol for total DNA purification from Gram-positive bacteria of the GRS genomic DNA kit – Bacteria -#GK07.0100 (Grisp, Porto, Portugal). PCR amplification of the 16S rRNA gene fragments was performed using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R ( 5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991) as described by VazMoreira et al. (2009). The 16S rRNA gene nucleotide sequences were used to query the EzBioCloud library (Yoon et al., 2017). 2.5. Antibiotic susceptibility testing The classification of each isolate in terms of their antibiotic susceptibility (sensitive, intermediate or resistant) was achieved according to the Clinical and Laboratory Standards Institute (CLSI, 2012). Listeria spp. isolate was classified as described by Barbosa et al. (2013). Antibiotics were chosen for each group of isolates according to their diverse representation of different classes of antimicrobial agents. The minimum inhibitory concentrations (MIC; μg/ml) were determined by ε-test for trimethoprim/sulphamethoxazole (SXT, AB Biodisk, Solna, Sweden) and by the agar dilution method for thirteen antibiotics. Each test was carried out on Muller-Hinton Agar (MHA, bioMérieux) with cations adjusted for penicillin G (Sigma, Steinheim, Germany) and ampicillin (Fluka, Steinheim, Germany) and on MHA for vancomycin (Fluka), oxacillin, ceftazidime, chloramphenicol, nalidixic acid, nitrofurantoin (Sigma), ciprofloxacin, erythromycin, gentamicin, tetracycline and rifampicin (all kindly supplied by the company Labesfal, Portugal). Each experiment was performed in duplicate and all isolates were grown on plates of MHA and MHA with cations adjusted with no added antibiotics as negative controls. The quality control strains Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used to monitor the accuracy of MICs (CLSI, 2012). Plates were incubated at 37 °C for 24 h. Isolates exhibiting resistance to, at least, two of the antimicrobial agents of different classes were considered to be multi-resistant strains. 2.5.1. Determination of mecA gene for staphylocci isolates According to CLSI (2012), “oxacillin interpretative criteria may overcall resistance for some coagulase-negative staphylococci, because some non-S. epidermidis strains for which the oxacillin MICs are 0.5–2 μg/ml lack mecA”. In this sense, all coagulase-negative staphylococci isolates with oxacillin MICs of 0.5–2 μg/mL were tested for the presence of mecA gene (CLSI, 2012). All the procedures were performed according to Castro, Santos, Meireles, Silva, and Teixeira (2016). 2.6. Statistical analysis Statistical analysis was performed with the IBM SPSS Statistics, 24 (IBM Corporation, USA). Differences between the visual appearance of the plastic bags (“very little use” or “extended use” and “apparently clean” or “dirty”) and the results of enumeration obtained were compared using the Student t-test. The mean difference was considered significant at the 0.05 level. 3. Results and discussion Results for enumeration and detection of different microorganisms for 30 used reusable plastic bags studied are presented in Table 1. Counts of total viable microorganisms, Enterobacteriaceae and enterococci were below the detection limit of the enumeration technique for the two unused reusable plastic bags used as control (data not shown). Only two samples showed high counts for total viable 160
Food Control 99 (2019) 158–163
J. Barbosa et al.
which will not undergo any treatment before being ingested. Between 31 isolates grown on bile esculin azide agar, and firstly mentioned as enterococci by esculin hydrolysis (Table 1), only 2 isolates were identified by 16s rRNA sequence as Enterococcus gallinarum. The remaining 29 isolates were identified as belonging to Staphylococcus spp. and other genera of lactic acid bacteria, such as Marinilactibacillus piezotolerans and Aerococcus urinaeequi. Marinilactibacillus piezotolerans was firstly isolated from deep sub-sea floor sediment by Toffin et al. (2005), but other species from this genera have been found in cheeses and spoiled dry cured-hams (Ishikawa et al., 2007; Rastelli, Giraffa, Carminati, Parolari, & Barbuti, 2005). Aerococcus urinaeequi are subsequently found in hospital environments and meat-curing brines and are very similar to enterococci (Rasmussen, 2016). The presence of the other organisms (lactic acid bacteria and staphylococci) is also not surprising, since some of these have been found in different foods, especially in fermented products of meat origin and cheeses (Afzal et al., 2010; Barbosa, Ferreira, & Teixeira, 2009; Fijałkowski, Peitler, & Karakulska, 2016; Pesavento, Calonico, Ducci, Magnanini, & Lo Nostro, 2014). The majority of the 32 isolates of Staphylococcus coagulase negative were identified as S. epidermidis (15). Most of the species identified have already been isolated from different ready-to-eat foods such as cheeses, cured meats, sausages and other fermented food products or smoked fish (Chajęcka-Wierzchowska, Zadernowska, Nalepa, Sierpińska, & Łaniewska-Trokenheim, 2015; Fijałkowski et al., 2016; Mainar et al., 2016; Place, Hiestand, Burri, & Teuber, 2002; Rodrigues et al., 2017). Since these bags are often reused, and potentially used for multiple purposes, the possibility of contamination by several food products as well as the consumer's hands exists and could explain the presence of some and varied staphylococci (Williams et al., 2011). Rusin, Maxwell, and Gerba (2002) studied the transference ability of Micrococcus luteus, Serratia rubidea and phage PRD-1 from fomites (initial inoculum of approximately 108 CFU/ml or PFU/ml) to hands and the subsequent transference from the fingertip (initial inoculum of approximately 106 CFU/ml or PFU/ml) to the lip. Although the authors had verified the highest transference rates from non-porous and hard surfaces, the numbers of bacteria transferred to the hands after handling porous surfaces were still very elevated (> 106 cells). From the fingertip to the lip, the authors observed a similar transference to the one that occurred from hard surfaces to hands (Rusin et al., 2002). The percentage of isolates (belonging to each group of bacteria) that were sensitive, intermediate and resistant to each tested antibiotics is shown in Table 2. Also distribution of percentage and number of each species resistant and intermediate resistant to different antibiotics are presented (Supplementary Table S2). Of the two isolates of Listeria spp. found, L. innocua (bag 2) was intermediate resistant to ciprofloxacin and SXT and L. monocytogenes (bag 20) was only resistant to erythromycin. Apart from this resistance, the pathogenic strain L. monocytogenes was sensitive to all antibiotics commonly used as first and second-choice therapy to treat listeriosis, as has also been observed by others (Maćkiw et al., 2016). None of the isolates belonging to Enterobacteriaceae family were resistant to gentamicin and ciprofloxacin. Resistances to nalidixic acid (bag 20), ceftazidime (bags 6, 18 and 30) and tetracycline (bag 16) were only observed for a small number of isolates. On the other hand, 38.4% and 12.3% of Enterobacteriaceae isolates were resistant (bags 2, 3, 6, 7, 9, 13, 15, 16, 18 and 23) and intermediate resistant (bags 2, 3, 9, 13, 20 and 24) to nitrofurantoin, respectively. Twenty three out of 73 isolates were multi-resistant (Table S2) and belong to samples 2, 3, 6, 7, 9, 16, 18, 20 and 24. In general, the percentage of Enterobacteriaceae isolates resistant to the antibiotics tested was low, but this should not be undervalued, due to the ability of some Enterobacteriaceae to acquire antibiotic resistances as well as virulence factors (Baylis et al., 2011). Only resistances to erythromycin, tetracycline, nitrofurantoin and rifampicin were found for lactic acid bacteria isolates. Erythromycin was the antibiotic with the highest number of resistant isolates (50%
Table 2 Percentage of sensitive, intermediate and resistant isolates to each tested antibiotics.
Listeria group (n = 2)
Enterobacteriaceae group (n = 73)
Ampicillin Penicillin Vancomycin Gentamycin Erithromycin Tetracycline Ciprofloxacin Nitrofurantoin Rifampicin Chloramphenicol SXT Ampicillin Ceftazidime Gentamicin Tetracycline Ciprofloxacin
Staphylococcus group (n = 47)
Lactic acid bacteria group (n = 16)
Nitrofurantoin Chloramphenicol Nalidixic acid Ampicillin Penicillin Oxacillin Ceftazidime Vancomycin Gentamicin Erithromycin Tetracycline Ciprofloxacin Ampicillin Penicillin Vancomycin Erithromycin Tetracycline Ciprofloxacin Nitrofurantoin Rifampicin Chloramphenicol
Sensitive isolates (n)
Intermediate isolates (n)
Resistant isolates (n)
100.0 (2) 100.0 (2) 100.0 (2) 100.0 (2) 50.0 (1) 100.0 (2) 50.0 (1) 100.0 (2) 100.0 (2) 100.0 (2) 50.0 (1) 69.9 (51) 94.5 (69) 100.0 (73) 93.2 (68) 100.0 (73) 49.3 (36) 76.7 (56) 98.6 (72) 46.8 (22) 42.6 (20) 61.7 (29) 55.3 (26) 100.0 (0) 100.0 (0) 36.2 (17) 85.1 (40) 97.9 (46) 100 (16) 100 (16) 100 (16) 37.5 (6) 93.8 (15) 100 (16) 50.0 (8) 75.0 (12) 100 (16)
0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 13.7 (10) 5.5 (4) 0.0 (0)
0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 16.4 (12) 0.0 (0) 0.0 (0)
6.8 (5) 0.0 (0)
0.0 (0) 0.0 (0)
12.3 (9) 17.8 (13) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 6.4 (3) 0.0 (0) 0.0 (0) 34.0 (16) 0.0 (0) 2.1 (1) 0.0 (0) 0.0 (0) 0.0 (0) 12.5 (2) 0.0 (0) 0.0 (0) 18.8 (3) 0.0 (0) 0.0 (0)
38.4 (28) 5.5 (4) 1.4 (1) 53.2 (25) 57.4 (27) 38.3 (18) 38.3 (18) 0.0 (0) 0.0 (0) 29.8 (14) 14.9 (7) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (8) 6.2 (1) 0.0 (0) 31.2 (4) 25.0 (4) 0.0 (0)
(Supplementary Table S1). Isolates of Listeria spp. detected were identified as L. innocua in plastic bag 2 and L. monocytogenes in plastic bag 20. The presence of the foodborne pathogen L. monocytogenes highlights the risk of cross-contamination, since this pathogen is normally found in ready-to-eat, fresh and non-processed food (Ferreira et al., 2006; Henriques & Fraqueza, 2017; Maćkiw et al., 2016). Most of the 73 Enterobacteriaceae isolates were identified as Pantoea spp. (Table S1). The bacterial genus Pantoea comprises many versatile species that have been isolated from a multitude of environments, such as aquatic and terrestrial environments, as well as in association with insects, animals and humans (Walterson & Stavrinides, 2015). All other genera found, such as Citrobacter spp., or Escherichia spp., are ubiquitously distributed in nature and have been isolated from food, water, and other environmental sources and in human clinical samples (Anahory, Darbas, Ongaro, Jean-Pierre, & Mion, 1998; Anuradha, 2014; Chart, 2012; Hoffmann et al., 2005; Leal-Negredo, Castelló-Abieta, Leiva, & Fernández, 2017; Wang et al., 2016). Many members of the Enterobacteriaceae family are responsible for spoilage of a variety of foods including fruits and vegetables, meats, poultry, eggs, milk and dairy products, as well as fish and other seafood (Baylis, Uyttendaele, Joosten, & Davies, 2011). Although there is a need to cook these raw foodstuffs thoroughly to avoid food poisoning outbreaks (Hennekinne, Herbin, Firmesse, & Auvray, 2015), the same does not happen with ready-to-eat products that are cross-contaminated with these products, 161
Food Control 99 (2019) 158–163
J. Barbosa et al.
resistant – bags 13 and 15 - and 12.5% intermediate resistant – bags 3, 5, 6, 12 and 13), followed by nitrofurantoin (31.2% resistant and 18.8% intermediate resistant – all from bag 13), rifampicin (25% resistant; bags 3 and 13) and, finally, tetracycline (6.2% resistant; bags 1, 13 and 16). Also multi-resistances were found, being all multi-resistant isolates from sample 13. Marinilactibacillus piezotolerans isolates were sensitive to all antibiotics tested, but on the other hand, three isolates of Aerococcus urinaeequi and all Enterococcus gallinarum (2) were multiresistant (Table S2). As previously described, aerococci share some features of antibiotic resistance with enterococci (reviewed by Rasmussen, 2016). Antibiotic resistance and/or multi-resistance of Enterococcus spp. isolated from different processed and ready-to-eat foods are well described (Barbosa et al., 2009; Pesavento et al., 2014). The ability of sensitive enterococci to acquire antibiotic resistances is a cause of concern, combined with the common presence of virulence factors in a high number of strains (Barbosa, Gibbs, & Teixeira, 2010). The highest percentages of isolates resistant to the different antibiotics tested were found for isolates belonging to the genus Staphylococcus. More than 50% of isolates were resistant to ampicillin and penicillin and, of those, 21% (10 isolates from bags 1, 3, 4, 5, 12, 13, 14, 15, 23 and 24) were simultaneously resistant to ampicillin, penicillin and oxacillin. All resistant isolates with oxacillin MICs of 0.5–2 μg/mL were tested for the presence of mecA gene. None of the isolates harbored the mecA gene (data not shown), indicating that none of these multi-resistant isolates were classified as resistant to methicillin. Also the number of isolates resistant to ceftazidime (38.3% resistant – bags 1–7, 11–16 and 23–25 - and 6.4% intermediate resistant – bags 1, 6 and 15) and erythromycin (29.8% resistant – bags 3, 12, 14, 18, 22, 23, 25–27, 29 and 30 - and 34.0% intermediate resistant – bags 1, 3–6, 11–13, 15, 17, 21, 24 and 27) was elevated. None of the isolates were resistant to vancomycin or gentamicin. However, only 10 isolates from 47 were not multi-resistant (Table S2; from bags 1, 3, 7, 9, 12 and 23). Other authors reported high percentages of antibiotic resistant and multi-resistant coagulase-negative staphylococci isolated from different foods (Fijałkowski et al., 2016; Nunes, Aguila, & Paschoalin, 2015) and despite that coagulase negative staphylococci are not classical food poisoning bacteria, their ability to spread antibiotic resistances to others of the same genus, as Staphylococcus aureus, or even to other pathogens, is significantly relevant. In summary, the use of reusable plastic bags for different purposes and the transport of different foods, carrying different microorganisms, can pose a problem of contamination, especially, cross-contamination. Using separate bags for different classes of products, as meats, fresh fruits and vegetables, and ready-to-eat foods, as well as their frequent washing could be a starting point to reduce cross-contamination and it is already recommended by Government Agency of Food Safety from USA (Gieraltowski, 2012).
Declarations of interest None. Acknowledgments This work was scientifically supported by National Funds from the Fundação para a Ciência e a Tecnologia (FCT, Portugal) through project UID/Multi/50016/2013 and through project “Biological tools for adding and defending value in key agro-food chains (bio – n2 – value)”, nº NORTE-01-0145-FEDER-000030, funded by Fundo Europeu de Desenvolvimento Regional (FEDER, Portugal), under Programa Operacional Regional do Norte - Norte2020”. Financial support for author J. Barbosa was provided by a post-doctoral fellowship SFRH/ BPD/113303/2015 (FCT, Portugal). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2018.12.041. References Afzal, M. I., Jacquet, T., Delaunay, S., Borges, F., Millière, J. B., Revol-Junelles, A. M., et al. (2010). Carnobacterium maltaromaticum: Identification, isolation tools, ecology and technological aspects in dairy products. Food Microbiology, 27, 573–579. https:// doi.org/10.1016/j.fm.2010.03.019. Anahory, T., Darbas, H., Ongaro, O., Jean-Pierre, H., & Mion, P. (1998). Serratia ficaria: a misidentified or unidentified rare cause of human infections in fig tree culture zones. Journal of Clinical Microbiology, 36, 3266–3272. Anuradha, M. (2014). Leclercia Adecarboxylata isolation: case reports and review. Journal of Clinical and Diagnostic Research, 8, DD03–DD04. https://doi.org/10.7860/JCDR/ 2014/9763.5260. Barbosa, J., Ferreira, V., & Teixeira, P. (2009). Antibiotic susceptibility of enterococci isolated from traditional fermented meat products. Food Microbiology, 26, 527–532. https://doi.org/10.1016/j.fm.2009.03.005. Barbosa, J., Gibbs, P. A., & Teixeira, P. (2010). Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal. Food Control, 21, 651–656. https://doi.org/10.1016/j.foodcont.2009.10.002. Barbosa, J., Magalhães, R., Santos, S., Ferreira, V., Brandão, T. R. S., Silva, J., et al. (2013). Evaluation of antibiotic resistance patterns of food and clinical Listeria monocytogenes isolates in Portugal. Foodborne Pathogens and Disease, 10, 861–866. https://doi.org/10.1089/fpd.2013.1532. Baylis, C., Uyttendaele, M., Joosten, H., & Davies, A. (2011). The Enterobacteriaceae and their significance to the food industry. ILSI Europe Report Series. Brussels, Belgiumwww. ilsi.eu. Bradford, M. A., Humphrey, T. J., & Lappin-Scott, H. M. (1997). The cross-contamination and survival of Salmonella enteritidis PT4 on sterile and non-sterile foodstuffs. Letters in Applied Microbiology, 24, 261–264. https://doi.org/10.1046/j.1472-765X.1997. 00127.x. Buchholz, A. L., Davidson, G. R., Marks, B. P., Todd, E. C. D., & Ryser, E. T. (2012). Transfer of Escherichia coli 0157:H7 from equipment surfaces to fresh-cut leafy greens during processing in a model pilot-plant production line with sanitizer-free water. Journal of Food Protection, 75, 1920–1929. https://doi.org/10.4315/0362-028X.JFP11-558. Byrd-Bredbenner, C., Berning, J., Martin-Biggers, J., & Quick, V. (2013). Food safety in home kitchens: a synthesis of the literature. International Journal of Environmental Research and Public Health, 10, 4060–4085. https://doi.org/10.3390/ ijerph10094060. Castro, A., Santos, C., Meireles, H., Silva, J., & Teixeira, P. (2016). Food handlers as potential sources of dissemination of virulent strains of Staphylococcus aureus in the community. Journal of Infection and Public Health, 9, 153–160. https://doi.org/10. 1016/j.jiph.2015.08.001. Chajęcka-Wierzchowska, W., Zadernowska, A., Nalepa, B., Sierpińska, M., & ŁaniewskaTrokenheim, Ł. (2015). Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin - phenotypic and genotypic antibiotic resistance. Food Microbiology, 46, 222–226. https://doi.org/10.1016/j.fm.2014.08.001. Chart, H. (2012). Klebsiella, Enterobacter, Proteus and other enterobacteria: Pneumonia; urinary tract infection; opportunist infection. Medical Microbiology (pp. 290–297). chapter 27. Clinical and Laboratory Standards Institute (2012). Performance Standards for Antimicrobial Susceptibility Tests; Document M100. Wayne, PA: Clinical and Laboratory Standards Institute. Ferreira, V., Barbosa, J., Vendeiro, S., Mota, A., Silva, F., Monteiro, M. J., et al. (2006). Chemical and microbiological characterization of alheira: A typical Portuguese fermented sausage with particular reference to factors relating to food safety. Meat Science, 73, 570–575. https://doi.org/10.1016/j.meatsci.2006.02.011. Fijałkowski, K., Peitler, D., & Karakulska, J. (2016). Staphylococci isolated from ready-toeat meat – Identification, antibiotic resistance and toxin gene profile. International
4. Conclusions In this study, we showed that the 30 analyzed reusable plastic bags were contaminated with different microorganisms, including pathogens. Among them, we also found several antibiotic resistant isolates, including multi-resistances. It was demonstrated that appearance is not a reliable datum about the bag contamination. Microbiological evaluation of the efficacy of simple washing/sanitising procedures applied in the domestic environment could be an interesting study to perform with contaminated reusable plastic bags. We believe that this study clearly demonstrates the need of education campaigns to alert the public about the misuse of their reusable plastic bags and how this may affect their health. The competent authorities should contribute to tentatively minimize this problem and one simple measure can start with printing instructions on reusable bags, alerting to the need for washing and separation of raw and readyto-eat foods. 162
Food Control 99 (2019) 158–163
J. Barbosa et al.
1016/j.wasman.2017.01.023. Nunes, R. S. C., Aguila, E. M. D., & Paschoalin, V. M. F. (2015). Safety evaluation of the coagulase negative staphylococci microbiota of salami: superantigenic toxin production and antimicrobial resistance. BioMed Research International, 483548. https:// doi.org/10.1155/2015/483548. Pesavento, G., Calonico, C., Ducci, B., Magnanini, A., & Lo Nostro, A. (2014). Prevalence and antibiotic resistance of Enterococcus spp. isolated from retail cheese, ready-to-eat salads, ham, and raw meat. Food Microbiology, 41, 1–7. https://doi.org/10.1016/j.fm. 2014.01.008. Place, R. B., Hiestand, D., Burri, S., & Teuber, M. (2002). Staphylococcus succinus subsp. casei subsp. nov., a dominant isolate from a surface ripened cheese. Systematic & Applied Microbiology, 25, 353–359. https://doi.org/10.1078/0723-2020-00130. Rasmussen, M. (2016). Aerococcus: an increasingly acknowledged human pathogen. Clinical Microbiology and Infections, 22, 22–27. https://doi.org/10.1016/j.cmi.2015. 09.026. Rastelli, E., Giraffa, G., Carminati, D., Parolari, G., & Barbuti, S. (2005). Identification and characterisation of halotolerant bacteria in spoiled dry-cured hams. Meat Science, 70, 241–246. https://doi.org/10.1016/j.meatsci.2005.01.008. Repp, K., & Keene, W. (2012). A point-source norovirus outbreak caused by exposure to fomites. The Journal of Infectious Diseases, 205, 1639–1641. https://doi.org/10.1093/ infdis/jis250. Rodrigues, M. X., Silva, N. C. C., Trevilin, J. H., Cruzado, M. M. B., Mui, T. S., Duarte, F. R. S., et al. (2017). Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants. Journal of Dairy Science, 100, 1–9. https://doi.org/10.3168/jds.2016-12477. Rusin, P., Maxwell, S., & Gerba, C. (2002). Comparative surface-to-hand and fingertip-tomouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage. Journal of Applied Microbiology, 93, 585–592. https://doi.org/10.1046/j.13652672.2002.01734.x. Summerbell, R. (2009). Grocery Carry Bag Sanitation, A Microbiological Study of Reusable Bags and ‘First or single-use’ Plastic Bags, Sporometrics, Toronto Canada. http://www. carrierbagtax.com/downloads/Microbiological_Study_of_Reusable_Grocery_Bags.pdf Acessed June 2018. Tabasco, R., Paarup, T., Janer, C., Peláez, C., & Requena, T. (2007). Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk. International Dairy Journal, 17, 1107–1114. Toffin, L., Zink, K., Kato, C., Pignet, P., Bidault, A., Bienvenu, N., et al. (2005). Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough. International Journal of Systematic and Evolutionary Microbiology, 55, 345–351. https://doi.org/10.1099/ijs.0. 63236-0. Vaz-Moreira, I., Faria, C., Lopes, A. R., Svensson, L., Falsen, E., Moore, E. R., et al. (2009). Sphingobium vermicomposti sp. nov., isolated from vermicompost. International Journal of Systematic and Evolutionary Microbiology, 59, 3145–3149. https://doi.org/10.1099/ ijs.0.006163-0. Walterson, A. M., & Stavrinides, J. (2015). Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiology Reviews, 39, 968–984. https://doi.org/10.1093/femsre/fuv027. Wang, Y., Jiang, X., Xu, Z., Ying, C., Yu, W., & Xiao, Y. (2016). Case report: Identification of Raoultella terrigena as a rare causative agent of subungual abscess based on 16S rRNA and housekeeping gene sequencing. The Canadian Journal of Infectious Diseases & Medical Microbiology3879635. 4 pages https://doi.org/10.1155/2016/3879635. Williams, D. L., Gerba, C. P., Maxwell, S., & Si, R. G. (2011). Assessment of the potential for cross-contamination of food products by reusable shopping bags. Food Protection Trends, 31, 508–513. Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., et al. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome bassemblies. International Journal of Systematic and Evolutionary Microbiology, 67, 1613–1617. https://doi.org/10.1099/ijsem.0.001755.
Journal of Food Microbiology, 238, 113–120. https://doi.org/10.1016/j.ijfoodmicro. 2016.09.001. Gieraltowski (2012). Reusable grocery bags: Keep ‘em clean while going green. https://www. foodsafety.gov/blog/reusable_bags.html Assessed June 2018. Hennekinne, J.-A., Herbin, S., Firmesse, O., & Auvray, F. (2015). European food poisoning outbreaks involving meat and meat-based products. International 58th Meat Industry Conference “Meat Safety and Quality: Where it goes?”. Procedia Food Science, 5, 93–96. Henriques, A. R., & Fraqueza, M. J. (2017). Biofilm-forming ability and biocide susceptibility of Listeria monocytogenes strains isolated from the ready-to-eat meat-based food products food chain. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 81, 180–187. https://doi.org/10.1016/j.lwt.2017.03.045. Hilton, A. (2015). Reusing plastic bags a 'contamination risk'. http://www.aston.ac.uk/ news/releases/2015/october-2015/reusing-plastic-bags-a-contamination-risk/, Accessed date: 2 October 2018. Hoffmann, H., Stindl, S., Stumpf, A., Mehlen, A., Monget, D., Heesemann, J., et al. (2005). Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Systematic & Applied Microbiology, 28, 206–212. https://doi.org/10.1016/j. syapm.2004.12.009. IPQ (1983). Food Microbiology. General rules for identifying coliform bacteria. Portuguese Standard NP 2164. IPQ (1986a). Food Microbiology. General rules for identifying Escherichia coli. Portuguese Standard NP 2308. IPQ (1986b). Food Microbiology. General rules for identifying Staphylococcus aureus. Portuguese Standard NP 2260. Ishikawa, M., Kodama, K., Yasuda, H., Okamoto-Kainuma, A., Koizumi, K., & Yamasato, K. (2007). Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses. Letters in Applied Microbiology, 44, 308–313. https://doi.org/10.1111/j. 1472-765X.2006.02073.x. ISO (1996). Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 1: Detection method. International Standard ISO 11290-1. ISO (2000). Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 2: colony-count method. International Standard ISO 21528-2. ISO (2013). Microbiology of the food chain - Horizontal method for the enumeration of microorganisms - Part 1: Colony count at 30 °C by the pour plate technique. International Standard ISO 4833-1. Kusumaningrum, H. D., Riboldi, G., Hazeleger, W. C., & Beumer, R. R. (2003). Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods. International Journal of Food Microbiology, 85, 227–236. https://doi.org/10.1016/ S0168-1605(02)00540-8. Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt, & M. Goodfellow (Eds.). Nucleic acid techniques in bacterial systematics (pp. 115–175). Chichester: Wiley. Leal-Negredo, A., Castelló-Abieta, C., Leiva, P. S., & Fernández, J. (2017). Urinary tract infection by Lelliottia amnigena (Enterobacter amnigenus): an uncommon pathogen. Revista Española de Quimioterapia, 30, 483–484. van Leeuwen, A. (2013). Bacterial and Viral Health Hazards of Reusable Shopping Bags. https://fighttheplasticbagban.files.wordpress.com/2014/10/bacterial-and-viralhealth-hazards-of-reusable-shopping-bags_rev_1.pdf Accessed June 2018. Maćkiw, E., Modzelewska, M., Mąka, L., Ścieżyńska, H., Pawłowska, K., Postupolski, J., et al. (2016). Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready-to-eat products in Poland in 2007-2011. Food Control, 59, 7–11. https://doi. org/10.1016/j.foodcont.2015.05.011. Mainar, M. S., Xhaferi, R., Samapundo, S., Simba, R., Frank, D., & Frédéric, L. (2016). Opportunities and limitations for the production of safe fermented meats without nitrate and nitrite using an antibacterial Staphylococcus sciuri starter culture. Food Control, 69, 267–274. https://doi.org/10.1016/j.foodcont.2016.04.056. Martinho, G., Balaia, N., & Pires, A. (2017). The Portuguese plastic carrier bag tax: The effects on consumers' behavior. Waste Management, 61, 3–12. https://doi.org/10.
163
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Microbiological contamination of reusable plastic bags for food transportation
T
J. Barbosaa, H. Albanoa, C.P. Silvaa,b, P. Teixeiraa,∗ a Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina, Laboratório Associado, Escola Superior de Biotecnologia, Rua Arquiteto Lobão Vital 172, 4200-374, Porto, Portugal b Colégio Internato dos Carvalhos, Rua do Padrão, 83, Carvalhos, 4415-284, Pedroso, Portugal
A R T I C LE I N FO
A B S T R A C T
Keywords: Domestic environment Foodborne pathogens Reusable plastic bag Cross-contamination Antibiotic resistance
Nowadays, with so many concerns for the environment, the use of reusable plastic bags is becoming routine, instead of the use of polluting single-use plastic bags. However, this is controversial in terms of food safety, since consumers transport many different foods, which could contaminate their bags and pose a risk to their health due to cross-contamination. This study aimed to detect or enumerate several indicators/pathogens from 30 used reusable plastic (polypropylene) bags and, to evaluate their antibiotic resistance profiles after identification by 16s rRNA of each isolated microorganism. Several genera of Enterobacteriaceae, coagulase-negative staphylococci and also Listeria monocytogenes were found in the reusable plastic bags analyzed. In general, high percentages of antibiotics resistance were found, highlighting the elevated occurrence of multi-resistant isolates of coagulase-negative staphylococci and Enterobacteriaceae. This study demonstrates the level and variety of microbial contamination of some used reusable plastic bags. No correlation was found between microbial levels and the visual appearance of each bag demonstrating that appearance is not a reliable datum about the bag contamination. We believe that this study could help the competent authorities taking measures to alert consumers to good food safety practices, not only in their kitchens, but also in the bags that carry their food.
1. Introduction Reusable plastic bags for transport of groceries from the store to the consumer's home have become popular in recent years (Williams, Gerba, Maxwell, & Si, 2011). In Portugal, this trend became more noticeable from February 2015, since plastic bags began to be taxed (Martinho, Balaia, & Pires, 2017). This additional cost resulted in an increase in the use of ‘bags for life’, i.e., resistant reusable plastic bags. However, “reusable” does not mean “clean”. Reusing plastic bags is beneficial to the environment, but as already stated “the public should be mindful of the ability of bacteria to contaminate and survive for long periods of time. Bacteria can easily transfer from different types of reusable bags to the hand and back again” (Hilton, 2015). In the U.S., reusable grocery bags are considered a new cross-contamination vehicle that has the potential to pose a significant risk of bacterial cross-contamination (Byrd-Bredbenner, Berning, Martin-Biggers, & Quick, 2013). Another important issue is that using the same bag for different purposes increases the risk of contaminating the bag with a whole host of bacteria. Understanding the importance of using different bags for
∗
different purposes is an important topic for consumers. However, it was reported that one in three consumers used these bags for more than just groceries, such as gym bags, toy bags, among other uses and that 75% of consumers use these same bags for carrying raw meat and other foods (Williams et al., 2011). In addition, the same authors stated that reusable bags, if not properly washed, could play a role in the cross-contamination of foods. Contaminated reusable grocery bags could pose a foodborne illness risk - an outbreak of norovirus in a girls' soccer team was traced to a contaminated reusable grocery bag (Repp & Keene, 2012). Considering that there are few studies regarding the microbial contamination of these plastic bags, the objective of this study was to evaluate the potential for reusable bags being contaminated. For that, several indicators/pathogens were detected or enumerated, subjected to 16S rRNA identification and their antibiotic resistance profiles assessed.
Corresponding author. E-mail address: [email protected] (P. Teixeira).
https://doi.org/10.1016/j.foodcont.2018.12.041 Received 25 October 2018; Received in revised form 27 December 2018; Accepted 28 December 2018 Available online 31 December 2018 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.
Food Control 99 (2019) 158–163
J. Barbosa et al.
Table 1 Enumeration (log CFU/bag) and detection (presence or absence/bag) data of several microorganisms for 30 used reusable plastic bags studied. Sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Area (cm2)
4838 4672 5424 4736 4928 4440 4636 4988 5031 3298 6078 5891 6078 6120 5990 4890 4890 4890 4890 4890 5301 5226 4538 4950 4928 4992 4736 5159 5226 4890
Enumeration (log CFU/bag)
Detection (presence or absence/bag)
Total microorganisms at 30 °C
Enterobacteriaceae
Enterococci
Coliforms 30 °C
E. coli
Staphylococcus coagulase -
Staphylococcus coagulase +
Listeria spp.
Listeria monocytogenes
4.8 3.4 6.3 3.5 3.9 3.5 3.6 3.9 4.3 2.8 2.5 3.4 5.9 3.2 3.2 7.3 2.4 3.7 2.4 3.5 2.4 1.9 4.9 3.0 2.4 2.0 2.5 2.2 2.5 3.9
< 1.0 3.4 4.7 < 1.0 < 1.0 3.9 2.9 < 1.0 3.9 < 1.0 < 1.0 < 1.0 5.2 3.5 3.0 4.5 < 1.0 2.8 < 1.0 < 1.0 2.8 < 1.0 < 1.0 3.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 2.7
2.7 2.0 2.8 2.0 2.5 5.7 2.9 < 2.0 < 2.0 < 2.0 < 2.0 2.0 5.1 < 2.0 2.3 5.2 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 3.7 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0 < 2.0
– – + – – + – – + – – – – + – + – – – – – – + – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
+ + + + + + + – + – + + + + + + + + – – + + + + + + + – + +
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– + – – – – – – – – – – – – – – – – – + – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – + – – – – – – – – – –
Legend: Presence (+) or absence (−)/bag.
2. Materials and methods
Enterobacteriaceae on violet red bile dextrose agar (VRBD, Merck) (ISO 21528-1: 2000) incubated at 37 °C for 48 h. Detection of Listeria spp. was performed in half Fraser broth (Merck), incubated at 30 °C for 24 h, followed by all confirmatory tests (ISO 11290-1: 1996). Detection of coagulase-positive Staphylococcus was performed according to a Portuguese Standard (NP 2260: 1986) in which 1 ml of each sample in BPW solution (Sampling section) was sown in simple Chapman broth (tryptone 5 gl-1; meat extract 6 gl-1; protease peptone 5 gl-1; NaCl 75 gl-1; lactose 7.5 gl-1; agar 0.5 gl-1) and incubated at 37 °C during 24 and 48 h. Cultures were then transferred to BairdParker Agar with egg yolk tellurite (BPA, Biokar Diagnostics) and plates incubated for 24–48 h at 37 °C; characteristic colonies were confirmed by coagulase test with rabbit plasma (bioMérieux, Marcy l’Etoile, France). Coliforms at 30 °C and Escherichia coli were detected according to NP 2164: 1983 and NP 2308: 1986, respectively. After incubation in simple lactose broth (Lab M) at 30 °C during 48 h, coliforms at 30 °C were detected by growth and gas production in brilliant green broth (Oxoid, Basingstoke, United Kingdom) incubated at 30 °C for 48 h, and E. coli was detected also by growth and gas production in brilliant green broth and by indole production on peptone water, both incubated at 44.5 °C for 48 h. Enumeration of total microorganisms at 30 °C, Enterobacteriaceae and enterococci were also performed for two unused plastic bags immediately after being purchased (control bags).
2.1. Sampling This study was carried out in the Metropolitan Area of Porto, Portugal, from October to December, 2015. Thirty used and two unused (control) reusable plastic bags were sampled. These bags were constituted by 100% polypropylene (type of reusable bags sold in most supermarkets), varying only in size (information about sampling area is presented in Table 1). Each of the 30 used bags belonged to a different consumer. All consumers reported that they had no criterion in the type of food transported, i.e., the analyzed bags transported all types of food, raw or processed. However, with the exception of some vegetables, all raw food (meat and fish) was inside individual packages (plastic bags or polystyrene trays with a plastic film cover). Before sampling all the bags were visually inspected by the same laboratory technician and classified as “very little use” or “extended use” and “apparently clean” or “dirty”. Samples were collected using one cotton swab moistened with sterile quarter strength Ringer's solution (Lab M, Bury, United Kingdom), which was scrubbed over all the interior area and re-suspended in 10 ml of Buffered Peptone Water (BPW, Merck, Darmstadt, Germany). All samples were transported to the laboratory in a refrigerated box and analyzed as soon as they arrived (within 24 h). 2.2. Microbiological analyses
2.3. Origin of isolates
Appropriate decimal dilutions were prepared in sterile Ringer's solution for microbial enumeration according to ISO Standards: total viable aerobic microorganisms at 30 °C on plate count agar (PCA, Pronadisa, Madrid, Spain) (ISO 4833-1: 2013) and enterococci on bile esculin azide agar (BEAA, Biokar Diagnostics, Beauvais, France) (Ferreira et al., 2006), both incubated at 30 °C for 72 h and
Colonies (about 10%) of each selective culture media were randomly selected from plates having between 15 and 150 colonies (Tabasco, Paarup, Janer, Peláez, & Requena, 2007).
159
Food Control 99 (2019) 158–163
J. Barbosa et al.
microorganisms: sample 3 with 6.3 log CFU/bag and sample 16 with 7.3 log CFU/bag. For most of the bags, counts of Enterobacteriaceae were below the detection limit of the enumeration technique. Nevertheless, counts between 3.0 and 5.0 log CFU/bag were found in a small number of samples. Also enterococci were detected on 26 samples at levels close to or below the detection limit of the enumeration technique (2.0 log CFU/bag) and only 4 samples had higher numbers (samples 6, 13 and 16 with values of ∼5.0 log CFU/bag and sample 23 with 3.0 log CFU/bag). It is important to highlight that all the inner surface of each bag was sampled, even areas which probably came into contact with food infrequently, in particular the upper areas in the larger bags. So, although results are reported per bag, these contaminants are probably concentrated in zones such as the bottom of the bags where the probability of cross-contamination to foods is higher. Each bag was classified taking into account its visual appearance at the time of sample collection (data not shown). No significant relation (P > 0.05) was found between the microbial load and the visual appearance of each bag, i.e. with “very little use” or “extended use” and “apparently clean” or “dirty”. No Staphylococcus coagulase positive were detected in any plastic bags. However, Staphylococcus coagulase negative were detected in 25 of the 30 bags analyzed. Although none of the samples revealed the presence of Escherichia coli, coliforms were detected in five bags (3, 9, 14, 16 and 23). Listeria spp. was detected in two bags and in one of them Listeria monocytogenes was present. Our results are in accordance with some of the few studies on the subject. Williams et al. (2011) found high numbers of bacteria (including fecal coliforms) in every reusable bag collected from consumers outside a grocery store. Summerbell (2009) tested 49 “used” reusable shopping bags and found that the majority had some bacterial counts, 30% elevated bacterial counts and 12% unacceptable coliform counts. Interesting to note that Summerbell (2009) and Williams et al. (2011) showed that no bacteria were found in single use plastic carryout bags or new reusable bags. Bags containing coliform bacteria could indicate that they were contaminated by raw meats or other uncooked food products; their presence demonstrates that bags become contaminated and that foodborne pathogens could exist on the bags (van Leeuwen, 2013). In fact, the major problem associated with the presence of foodborne pathogens in reusable bags is the increased risk of bacterial cross-contamination. Already in 1997, Bradford, Humphrey, & LappinScott investigated the ability of two strains of Salmonella Enteritidis PT4 to cross-contaminate from inoculated egg droplets on surfaces onto melon or beef. The authors found that cross-contamination in each portion of food occurred in 1 s, when placed on the surfaces where the egg drops were wet, and up to 1 min when the egg droplets were dry (Bradford, Humphrey, & Lappin-Scott, 1997). Also Buchholz, Davidson, Marks, Todd, and Ryser (2012) demonstrated the occurrence of crosscontamination of E. coli O157:H7, even in very low levels, between fresh-cut leafy greens. The authors proved that E. coli O157:H7 from one contaminated batch of leafy greens could easily be spread to subsequent batches of uncontaminated product in a processing facility (Buchholz et al., 2012). Despite the steel stainless be of a different material from the reusable bags, the study of Kusumaningrum, Riboldi, Hazeleger, and Beumer (2003) is no less important. The authors studied the transference of Salmonella Enteritidis, Staphylococcus aureus and Campylobacter jejuni, at different initial levels, from kitchen sponges to stainless steel surfaces and from these to slices of cucumber and roasted chicken fillet. Cross-contamination of all microorganisms occurred from surfaces to food, with or without pressure applied, with transference rates varying from 50% to more than 100% for transmission to cucumber slices, and from 25% to 100% for transmission to roasted chicken fillet slices (Kusumaningrum et al., 2003). Microorganisms obtained from the enumeration and/or detection (Table 1) were isolated and identified by 16s rRNA sequence
2.4. LAB identification by 16S rRNA sequencing Deoxyribonucleic acid (DNA) of the isolates was extracted according to the protocol for total DNA purification from Gram-positive bacteria of the GRS genomic DNA kit – Bacteria -#GK07.0100 (Grisp, Porto, Portugal). PCR amplification of the 16S rRNA gene fragments was performed using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R ( 5′-GGTTACCTTGTTACGACTT-3′) (Lane, 1991) as described by VazMoreira et al. (2009). The 16S rRNA gene nucleotide sequences were used to query the EzBioCloud library (Yoon et al., 2017). 2.5. Antibiotic susceptibility testing The classification of each isolate in terms of their antibiotic susceptibility (sensitive, intermediate or resistant) was achieved according to the Clinical and Laboratory Standards Institute (CLSI, 2012). Listeria spp. isolate was classified as described by Barbosa et al. (2013). Antibiotics were chosen for each group of isolates according to their diverse representation of different classes of antimicrobial agents. The minimum inhibitory concentrations (MIC; μg/ml) were determined by ε-test for trimethoprim/sulphamethoxazole (SXT, AB Biodisk, Solna, Sweden) and by the agar dilution method for thirteen antibiotics. Each test was carried out on Muller-Hinton Agar (MHA, bioMérieux) with cations adjusted for penicillin G (Sigma, Steinheim, Germany) and ampicillin (Fluka, Steinheim, Germany) and on MHA for vancomycin (Fluka), oxacillin, ceftazidime, chloramphenicol, nalidixic acid, nitrofurantoin (Sigma), ciprofloxacin, erythromycin, gentamicin, tetracycline and rifampicin (all kindly supplied by the company Labesfal, Portugal). Each experiment was performed in duplicate and all isolates were grown on plates of MHA and MHA with cations adjusted with no added antibiotics as negative controls. The quality control strains Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used to monitor the accuracy of MICs (CLSI, 2012). Plates were incubated at 37 °C for 24 h. Isolates exhibiting resistance to, at least, two of the antimicrobial agents of different classes were considered to be multi-resistant strains. 2.5.1. Determination of mecA gene for staphylocci isolates According to CLSI (2012), “oxacillin interpretative criteria may overcall resistance for some coagulase-negative staphylococci, because some non-S. epidermidis strains for which the oxacillin MICs are 0.5–2 μg/ml lack mecA”. In this sense, all coagulase-negative staphylococci isolates with oxacillin MICs of 0.5–2 μg/mL were tested for the presence of mecA gene (CLSI, 2012). All the procedures were performed according to Castro, Santos, Meireles, Silva, and Teixeira (2016). 2.6. Statistical analysis Statistical analysis was performed with the IBM SPSS Statistics, 24 (IBM Corporation, USA). Differences between the visual appearance of the plastic bags (“very little use” or “extended use” and “apparently clean” or “dirty”) and the results of enumeration obtained were compared using the Student t-test. The mean difference was considered significant at the 0.05 level. 3. Results and discussion Results for enumeration and detection of different microorganisms for 30 used reusable plastic bags studied are presented in Table 1. Counts of total viable microorganisms, Enterobacteriaceae and enterococci were below the detection limit of the enumeration technique for the two unused reusable plastic bags used as control (data not shown). Only two samples showed high counts for total viable 160
Food Control 99 (2019) 158–163
J. Barbosa et al.
which will not undergo any treatment before being ingested. Between 31 isolates grown on bile esculin azide agar, and firstly mentioned as enterococci by esculin hydrolysis (Table 1), only 2 isolates were identified by 16s rRNA sequence as Enterococcus gallinarum. The remaining 29 isolates were identified as belonging to Staphylococcus spp. and other genera of lactic acid bacteria, such as Marinilactibacillus piezotolerans and Aerococcus urinaeequi. Marinilactibacillus piezotolerans was firstly isolated from deep sub-sea floor sediment by Toffin et al. (2005), but other species from this genera have been found in cheeses and spoiled dry cured-hams (Ishikawa et al., 2007; Rastelli, Giraffa, Carminati, Parolari, & Barbuti, 2005). Aerococcus urinaeequi are subsequently found in hospital environments and meat-curing brines and are very similar to enterococci (Rasmussen, 2016). The presence of the other organisms (lactic acid bacteria and staphylococci) is also not surprising, since some of these have been found in different foods, especially in fermented products of meat origin and cheeses (Afzal et al., 2010; Barbosa, Ferreira, & Teixeira, 2009; Fijałkowski, Peitler, & Karakulska, 2016; Pesavento, Calonico, Ducci, Magnanini, & Lo Nostro, 2014). The majority of the 32 isolates of Staphylococcus coagulase negative were identified as S. epidermidis (15). Most of the species identified have already been isolated from different ready-to-eat foods such as cheeses, cured meats, sausages and other fermented food products or smoked fish (Chajęcka-Wierzchowska, Zadernowska, Nalepa, Sierpińska, & Łaniewska-Trokenheim, 2015; Fijałkowski et al., 2016; Mainar et al., 2016; Place, Hiestand, Burri, & Teuber, 2002; Rodrigues et al., 2017). Since these bags are often reused, and potentially used for multiple purposes, the possibility of contamination by several food products as well as the consumer's hands exists and could explain the presence of some and varied staphylococci (Williams et al., 2011). Rusin, Maxwell, and Gerba (2002) studied the transference ability of Micrococcus luteus, Serratia rubidea and phage PRD-1 from fomites (initial inoculum of approximately 108 CFU/ml or PFU/ml) to hands and the subsequent transference from the fingertip (initial inoculum of approximately 106 CFU/ml or PFU/ml) to the lip. Although the authors had verified the highest transference rates from non-porous and hard surfaces, the numbers of bacteria transferred to the hands after handling porous surfaces were still very elevated (> 106 cells). From the fingertip to the lip, the authors observed a similar transference to the one that occurred from hard surfaces to hands (Rusin et al., 2002). The percentage of isolates (belonging to each group of bacteria) that were sensitive, intermediate and resistant to each tested antibiotics is shown in Table 2. Also distribution of percentage and number of each species resistant and intermediate resistant to different antibiotics are presented (Supplementary Table S2). Of the two isolates of Listeria spp. found, L. innocua (bag 2) was intermediate resistant to ciprofloxacin and SXT and L. monocytogenes (bag 20) was only resistant to erythromycin. Apart from this resistance, the pathogenic strain L. monocytogenes was sensitive to all antibiotics commonly used as first and second-choice therapy to treat listeriosis, as has also been observed by others (Maćkiw et al., 2016). None of the isolates belonging to Enterobacteriaceae family were resistant to gentamicin and ciprofloxacin. Resistances to nalidixic acid (bag 20), ceftazidime (bags 6, 18 and 30) and tetracycline (bag 16) were only observed for a small number of isolates. On the other hand, 38.4% and 12.3% of Enterobacteriaceae isolates were resistant (bags 2, 3, 6, 7, 9, 13, 15, 16, 18 and 23) and intermediate resistant (bags 2, 3, 9, 13, 20 and 24) to nitrofurantoin, respectively. Twenty three out of 73 isolates were multi-resistant (Table S2) and belong to samples 2, 3, 6, 7, 9, 16, 18, 20 and 24. In general, the percentage of Enterobacteriaceae isolates resistant to the antibiotics tested was low, but this should not be undervalued, due to the ability of some Enterobacteriaceae to acquire antibiotic resistances as well as virulence factors (Baylis et al., 2011). Only resistances to erythromycin, tetracycline, nitrofurantoin and rifampicin were found for lactic acid bacteria isolates. Erythromycin was the antibiotic with the highest number of resistant isolates (50%
Table 2 Percentage of sensitive, intermediate and resistant isolates to each tested antibiotics.
Listeria group (n = 2)
Enterobacteriaceae group (n = 73)
Ampicillin Penicillin Vancomycin Gentamycin Erithromycin Tetracycline Ciprofloxacin Nitrofurantoin Rifampicin Chloramphenicol SXT Ampicillin Ceftazidime Gentamicin Tetracycline Ciprofloxacin
Staphylococcus group (n = 47)
Lactic acid bacteria group (n = 16)
Nitrofurantoin Chloramphenicol Nalidixic acid Ampicillin Penicillin Oxacillin Ceftazidime Vancomycin Gentamicin Erithromycin Tetracycline Ciprofloxacin Ampicillin Penicillin Vancomycin Erithromycin Tetracycline Ciprofloxacin Nitrofurantoin Rifampicin Chloramphenicol
Sensitive isolates (n)
Intermediate isolates (n)
Resistant isolates (n)
100.0 (2) 100.0 (2) 100.0 (2) 100.0 (2) 50.0 (1) 100.0 (2) 50.0 (1) 100.0 (2) 100.0 (2) 100.0 (2) 50.0 (1) 69.9 (51) 94.5 (69) 100.0 (73) 93.2 (68) 100.0 (73) 49.3 (36) 76.7 (56) 98.6 (72) 46.8 (22) 42.6 (20) 61.7 (29) 55.3 (26) 100.0 (0) 100.0 (0) 36.2 (17) 85.1 (40) 97.9 (46) 100 (16) 100 (16) 100 (16) 37.5 (6) 93.8 (15) 100 (16) 50.0 (8) 75.0 (12) 100 (16)
0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 13.7 (10) 5.5 (4) 0.0 (0)
0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (1) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 16.4 (12) 0.0 (0) 0.0 (0)
6.8 (5) 0.0 (0)
0.0 (0) 0.0 (0)
12.3 (9) 17.8 (13) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 6.4 (3) 0.0 (0) 0.0 (0) 34.0 (16) 0.0 (0) 2.1 (1) 0.0 (0) 0.0 (0) 0.0 (0) 12.5 (2) 0.0 (0) 0.0 (0) 18.8 (3) 0.0 (0) 0.0 (0)
38.4 (28) 5.5 (4) 1.4 (1) 53.2 (25) 57.4 (27) 38.3 (18) 38.3 (18) 0.0 (0) 0.0 (0) 29.8 (14) 14.9 (7) 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 50.0 (8) 6.2 (1) 0.0 (0) 31.2 (4) 25.0 (4) 0.0 (0)
(Supplementary Table S1). Isolates of Listeria spp. detected were identified as L. innocua in plastic bag 2 and L. monocytogenes in plastic bag 20. The presence of the foodborne pathogen L. monocytogenes highlights the risk of cross-contamination, since this pathogen is normally found in ready-to-eat, fresh and non-processed food (Ferreira et al., 2006; Henriques & Fraqueza, 2017; Maćkiw et al., 2016). Most of the 73 Enterobacteriaceae isolates were identified as Pantoea spp. (Table S1). The bacterial genus Pantoea comprises many versatile species that have been isolated from a multitude of environments, such as aquatic and terrestrial environments, as well as in association with insects, animals and humans (Walterson & Stavrinides, 2015). All other genera found, such as Citrobacter spp., or Escherichia spp., are ubiquitously distributed in nature and have been isolated from food, water, and other environmental sources and in human clinical samples (Anahory, Darbas, Ongaro, Jean-Pierre, & Mion, 1998; Anuradha, 2014; Chart, 2012; Hoffmann et al., 2005; Leal-Negredo, Castelló-Abieta, Leiva, & Fernández, 2017; Wang et al., 2016). Many members of the Enterobacteriaceae family are responsible for spoilage of a variety of foods including fruits and vegetables, meats, poultry, eggs, milk and dairy products, as well as fish and other seafood (Baylis, Uyttendaele, Joosten, & Davies, 2011). Although there is a need to cook these raw foodstuffs thoroughly to avoid food poisoning outbreaks (Hennekinne, Herbin, Firmesse, & Auvray, 2015), the same does not happen with ready-to-eat products that are cross-contaminated with these products, 161
Food Control 99 (2019) 158–163
J. Barbosa et al.
resistant – bags 13 and 15 - and 12.5% intermediate resistant – bags 3, 5, 6, 12 and 13), followed by nitrofurantoin (31.2% resistant and 18.8% intermediate resistant – all from bag 13), rifampicin (25% resistant; bags 3 and 13) and, finally, tetracycline (6.2% resistant; bags 1, 13 and 16). Also multi-resistances were found, being all multi-resistant isolates from sample 13. Marinilactibacillus piezotolerans isolates were sensitive to all antibiotics tested, but on the other hand, three isolates of Aerococcus urinaeequi and all Enterococcus gallinarum (2) were multiresistant (Table S2). As previously described, aerococci share some features of antibiotic resistance with enterococci (reviewed by Rasmussen, 2016). Antibiotic resistance and/or multi-resistance of Enterococcus spp. isolated from different processed and ready-to-eat foods are well described (Barbosa et al., 2009; Pesavento et al., 2014). The ability of sensitive enterococci to acquire antibiotic resistances is a cause of concern, combined with the common presence of virulence factors in a high number of strains (Barbosa, Gibbs, & Teixeira, 2010). The highest percentages of isolates resistant to the different antibiotics tested were found for isolates belonging to the genus Staphylococcus. More than 50% of isolates were resistant to ampicillin and penicillin and, of those, 21% (10 isolates from bags 1, 3, 4, 5, 12, 13, 14, 15, 23 and 24) were simultaneously resistant to ampicillin, penicillin and oxacillin. All resistant isolates with oxacillin MICs of 0.5–2 μg/mL were tested for the presence of mecA gene. None of the isolates harbored the mecA gene (data not shown), indicating that none of these multi-resistant isolates were classified as resistant to methicillin. Also the number of isolates resistant to ceftazidime (38.3% resistant – bags 1–7, 11–16 and 23–25 - and 6.4% intermediate resistant – bags 1, 6 and 15) and erythromycin (29.8% resistant – bags 3, 12, 14, 18, 22, 23, 25–27, 29 and 30 - and 34.0% intermediate resistant – bags 1, 3–6, 11–13, 15, 17, 21, 24 and 27) was elevated. None of the isolates were resistant to vancomycin or gentamicin. However, only 10 isolates from 47 were not multi-resistant (Table S2; from bags 1, 3, 7, 9, 12 and 23). Other authors reported high percentages of antibiotic resistant and multi-resistant coagulase-negative staphylococci isolated from different foods (Fijałkowski et al., 2016; Nunes, Aguila, & Paschoalin, 2015) and despite that coagulase negative staphylococci are not classical food poisoning bacteria, their ability to spread antibiotic resistances to others of the same genus, as Staphylococcus aureus, or even to other pathogens, is significantly relevant. In summary, the use of reusable plastic bags for different purposes and the transport of different foods, carrying different microorganisms, can pose a problem of contamination, especially, cross-contamination. Using separate bags for different classes of products, as meats, fresh fruits and vegetables, and ready-to-eat foods, as well as their frequent washing could be a starting point to reduce cross-contamination and it is already recommended by Government Agency of Food Safety from USA (Gieraltowski, 2012).
Declarations of interest None. Acknowledgments This work was scientifically supported by National Funds from the Fundação para a Ciência e a Tecnologia (FCT, Portugal) through project UID/Multi/50016/2013 and through project “Biological tools for adding and defending value in key agro-food chains (bio – n2 – value)”, nº NORTE-01-0145-FEDER-000030, funded by Fundo Europeu de Desenvolvimento Regional (FEDER, Portugal), under Programa Operacional Regional do Norte - Norte2020”. Financial support for author J. Barbosa was provided by a post-doctoral fellowship SFRH/ BPD/113303/2015 (FCT, Portugal). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodcont.2018.12.041. References Afzal, M. I., Jacquet, T., Delaunay, S., Borges, F., Millière, J. B., Revol-Junelles, A. M., et al. (2010). Carnobacterium maltaromaticum: Identification, isolation tools, ecology and technological aspects in dairy products. Food Microbiology, 27, 573–579. https:// doi.org/10.1016/j.fm.2010.03.019. Anahory, T., Darbas, H., Ongaro, O., Jean-Pierre, H., & Mion, P. (1998). Serratia ficaria: a misidentified or unidentified rare cause of human infections in fig tree culture zones. Journal of Clinical Microbiology, 36, 3266–3272. Anuradha, M. (2014). Leclercia Adecarboxylata isolation: case reports and review. Journal of Clinical and Diagnostic Research, 8, DD03–DD04. https://doi.org/10.7860/JCDR/ 2014/9763.5260. Barbosa, J., Ferreira, V., & Teixeira, P. (2009). Antibiotic susceptibility of enterococci isolated from traditional fermented meat products. Food Microbiology, 26, 527–532. https://doi.org/10.1016/j.fm.2009.03.005. Barbosa, J., Gibbs, P. A., & Teixeira, P. (2010). Virulence factors among enterococci isolated from traditional fermented meat products produced in the North of Portugal. Food Control, 21, 651–656. https://doi.org/10.1016/j.foodcont.2009.10.002. Barbosa, J., Magalhães, R., Santos, S., Ferreira, V., Brandão, T. R. S., Silva, J., et al. (2013). Evaluation of antibiotic resistance patterns of food and clinical Listeria monocytogenes isolates in Portugal. Foodborne Pathogens and Disease, 10, 861–866. https://doi.org/10.1089/fpd.2013.1532. Baylis, C., Uyttendaele, M., Joosten, H., & Davies, A. (2011). The Enterobacteriaceae and their significance to the food industry. ILSI Europe Report Series. Brussels, Belgiumwww. ilsi.eu. Bradford, M. A., Humphrey, T. J., & Lappin-Scott, H. M. (1997). The cross-contamination and survival of Salmonella enteritidis PT4 on sterile and non-sterile foodstuffs. Letters in Applied Microbiology, 24, 261–264. https://doi.org/10.1046/j.1472-765X.1997. 00127.x. Buchholz, A. L., Davidson, G. R., Marks, B. P., Todd, E. C. D., & Ryser, E. T. (2012). Transfer of Escherichia coli 0157:H7 from equipment surfaces to fresh-cut leafy greens during processing in a model pilot-plant production line with sanitizer-free water. Journal of Food Protection, 75, 1920–1929. https://doi.org/10.4315/0362-028X.JFP11-558. Byrd-Bredbenner, C., Berning, J., Martin-Biggers, J., & Quick, V. (2013). Food safety in home kitchens: a synthesis of the literature. International Journal of Environmental Research and Public Health, 10, 4060–4085. https://doi.org/10.3390/ ijerph10094060. Castro, A., Santos, C., Meireles, H., Silva, J., & Teixeira, P. (2016). Food handlers as potential sources of dissemination of virulent strains of Staphylococcus aureus in the community. Journal of Infection and Public Health, 9, 153–160. https://doi.org/10. 1016/j.jiph.2015.08.001. Chajęcka-Wierzchowska, W., Zadernowska, A., Nalepa, B., Sierpińska, M., & ŁaniewskaTrokenheim, Ł. (2015). Coagulase-negative staphylococci (CoNS) isolated from ready-to-eat food of animal origin - phenotypic and genotypic antibiotic resistance. Food Microbiology, 46, 222–226. https://doi.org/10.1016/j.fm.2014.08.001. Chart, H. (2012). Klebsiella, Enterobacter, Proteus and other enterobacteria: Pneumonia; urinary tract infection; opportunist infection. Medical Microbiology (pp. 290–297). chapter 27. Clinical and Laboratory Standards Institute (2012). Performance Standards for Antimicrobial Susceptibility Tests; Document M100. Wayne, PA: Clinical and Laboratory Standards Institute. Ferreira, V., Barbosa, J., Vendeiro, S., Mota, A., Silva, F., Monteiro, M. J., et al. (2006). Chemical and microbiological characterization of alheira: A typical Portuguese fermented sausage with particular reference to factors relating to food safety. Meat Science, 73, 570–575. https://doi.org/10.1016/j.meatsci.2006.02.011. Fijałkowski, K., Peitler, D., & Karakulska, J. (2016). Staphylococci isolated from ready-toeat meat – Identification, antibiotic resistance and toxin gene profile. International
4. Conclusions In this study, we showed that the 30 analyzed reusable plastic bags were contaminated with different microorganisms, including pathogens. Among them, we also found several antibiotic resistant isolates, including multi-resistances. It was demonstrated that appearance is not a reliable datum about the bag contamination. Microbiological evaluation of the efficacy of simple washing/sanitising procedures applied in the domestic environment could be an interesting study to perform with contaminated reusable plastic bags. We believe that this study clearly demonstrates the need of education campaigns to alert the public about the misuse of their reusable plastic bags and how this may affect their health. The competent authorities should contribute to tentatively minimize this problem and one simple measure can start with printing instructions on reusable bags, alerting to the need for washing and separation of raw and readyto-eat foods. 162
Food Control 99 (2019) 158–163
J. Barbosa et al.
1016/j.wasman.2017.01.023. Nunes, R. S. C., Aguila, E. M. D., & Paschoalin, V. M. F. (2015). Safety evaluation of the coagulase negative staphylococci microbiota of salami: superantigenic toxin production and antimicrobial resistance. BioMed Research International, 483548. https:// doi.org/10.1155/2015/483548. Pesavento, G., Calonico, C., Ducci, B., Magnanini, A., & Lo Nostro, A. (2014). Prevalence and antibiotic resistance of Enterococcus spp. isolated from retail cheese, ready-to-eat salads, ham, and raw meat. Food Microbiology, 41, 1–7. https://doi.org/10.1016/j.fm. 2014.01.008. Place, R. B., Hiestand, D., Burri, S., & Teuber, M. (2002). Staphylococcus succinus subsp. casei subsp. nov., a dominant isolate from a surface ripened cheese. Systematic & Applied Microbiology, 25, 353–359. https://doi.org/10.1078/0723-2020-00130. Rasmussen, M. (2016). Aerococcus: an increasingly acknowledged human pathogen. Clinical Microbiology and Infections, 22, 22–27. https://doi.org/10.1016/j.cmi.2015. 09.026. Rastelli, E., Giraffa, G., Carminati, D., Parolari, G., & Barbuti, S. (2005). Identification and characterisation of halotolerant bacteria in spoiled dry-cured hams. Meat Science, 70, 241–246. https://doi.org/10.1016/j.meatsci.2005.01.008. Repp, K., & Keene, W. (2012). A point-source norovirus outbreak caused by exposure to fomites. The Journal of Infectious Diseases, 205, 1639–1641. https://doi.org/10.1093/ infdis/jis250. Rodrigues, M. X., Silva, N. C. C., Trevilin, J. H., Cruzado, M. M. B., Mui, T. S., Duarte, F. R. S., et al. (2017). Molecular characterization and antibiotic resistance of Staphylococcus spp. isolated from cheese processing plants. Journal of Dairy Science, 100, 1–9. https://doi.org/10.3168/jds.2016-12477. Rusin, P., Maxwell, S., & Gerba, C. (2002). Comparative surface-to-hand and fingertip-tomouth transfer efficiency of gram-positive bacteria, gram-negative bacteria, and phage. Journal of Applied Microbiology, 93, 585–592. https://doi.org/10.1046/j.13652672.2002.01734.x. Summerbell, R. (2009). Grocery Carry Bag Sanitation, A Microbiological Study of Reusable Bags and ‘First or single-use’ Plastic Bags, Sporometrics, Toronto Canada. http://www. carrierbagtax.com/downloads/Microbiological_Study_of_Reusable_Grocery_Bags.pdf Acessed June 2018. Tabasco, R., Paarup, T., Janer, C., Peláez, C., & Requena, T. (2007). Selective enumeration and identification of mixed cultures of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. acidophilus, L. paracasei subsp. paracasei and Bifidobacterium lactis in fermented milk. International Dairy Journal, 17, 1107–1114. Toffin, L., Zink, K., Kato, C., Pignet, P., Bidault, A., Bienvenu, N., et al. (2005). Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough. International Journal of Systematic and Evolutionary Microbiology, 55, 345–351. https://doi.org/10.1099/ijs.0. 63236-0. Vaz-Moreira, I., Faria, C., Lopes, A. R., Svensson, L., Falsen, E., Moore, E. R., et al. (2009). Sphingobium vermicomposti sp. nov., isolated from vermicompost. International Journal of Systematic and Evolutionary Microbiology, 59, 3145–3149. https://doi.org/10.1099/ ijs.0.006163-0. Walterson, A. M., & Stavrinides, J. (2015). Pantoea: insights into a highly versatile and diverse genus within the Enterobacteriaceae. FEMS Microbiology Reviews, 39, 968–984. https://doi.org/10.1093/femsre/fuv027. Wang, Y., Jiang, X., Xu, Z., Ying, C., Yu, W., & Xiao, Y. (2016). Case report: Identification of Raoultella terrigena as a rare causative agent of subungual abscess based on 16S rRNA and housekeeping gene sequencing. The Canadian Journal of Infectious Diseases & Medical Microbiology3879635. 4 pages https://doi.org/10.1155/2016/3879635. Williams, D. L., Gerba, C. P., Maxwell, S., & Si, R. G. (2011). Assessment of the potential for cross-contamination of food products by reusable shopping bags. Food Protection Trends, 31, 508–513. Yoon, S. H., Ha, S. M., Kwon, S., Lim, J., Kim, Y., Seo, H., et al. (2017). Introducing EzBioCloud: A taxonomically united database of 16S rRNA and whole genome bassemblies. International Journal of Systematic and Evolutionary Microbiology, 67, 1613–1617. https://doi.org/10.1099/ijsem.0.001755.
Journal of Food Microbiology, 238, 113–120. https://doi.org/10.1016/j.ijfoodmicro. 2016.09.001. Gieraltowski (2012). Reusable grocery bags: Keep ‘em clean while going green. https://www. foodsafety.gov/blog/reusable_bags.html Assessed June 2018. Hennekinne, J.-A., Herbin, S., Firmesse, O., & Auvray, F. (2015). European food poisoning outbreaks involving meat and meat-based products. International 58th Meat Industry Conference “Meat Safety and Quality: Where it goes?”. Procedia Food Science, 5, 93–96. Henriques, A. R., & Fraqueza, M. J. (2017). Biofilm-forming ability and biocide susceptibility of Listeria monocytogenes strains isolated from the ready-to-eat meat-based food products food chain. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 81, 180–187. https://doi.org/10.1016/j.lwt.2017.03.045. Hilton, A. (2015). Reusing plastic bags a 'contamination risk'. http://www.aston.ac.uk/ news/releases/2015/october-2015/reusing-plastic-bags-a-contamination-risk/, Accessed date: 2 October 2018. Hoffmann, H., Stindl, S., Stumpf, A., Mehlen, A., Monget, D., Heesemann, J., et al. (2005). Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Systematic & Applied Microbiology, 28, 206–212. https://doi.org/10.1016/j. syapm.2004.12.009. IPQ (1983). Food Microbiology. General rules for identifying coliform bacteria. Portuguese Standard NP 2164. IPQ (1986a). Food Microbiology. General rules for identifying Escherichia coli. Portuguese Standard NP 2308. IPQ (1986b). Food Microbiology. General rules for identifying Staphylococcus aureus. Portuguese Standard NP 2260. Ishikawa, M., Kodama, K., Yasuda, H., Okamoto-Kainuma, A., Koizumi, K., & Yamasato, K. (2007). Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses. Letters in Applied Microbiology, 44, 308–313. https://doi.org/10.1111/j. 1472-765X.2006.02073.x. ISO (1996). Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 1: Detection method. International Standard ISO 11290-1. ISO (2000). Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Enterobacteriaceae - Part 2: colony-count method. International Standard ISO 21528-2. ISO (2013). Microbiology of the food chain - Horizontal method for the enumeration of microorganisms - Part 1: Colony count at 30 °C by the pour plate technique. International Standard ISO 4833-1. Kusumaningrum, H. D., Riboldi, G., Hazeleger, W. C., & Beumer, R. R. (2003). Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods. International Journal of Food Microbiology, 85, 227–236. https://doi.org/10.1016/ S0168-1605(02)00540-8. Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt, & M. Goodfellow (Eds.). Nucleic acid techniques in bacterial systematics (pp. 115–175). Chichester: Wiley. Leal-Negredo, A., Castelló-Abieta, C., Leiva, P. S., & Fernández, J. (2017). Urinary tract infection by Lelliottia amnigena (Enterobacter amnigenus): an uncommon pathogen. Revista Española de Quimioterapia, 30, 483–484. van Leeuwen, A. (2013). Bacterial and Viral Health Hazards of Reusable Shopping Bags. https://fighttheplasticbagban.files.wordpress.com/2014/10/bacterial-and-viralhealth-hazards-of-reusable-shopping-bags_rev_1.pdf Accessed June 2018. Maćkiw, E., Modzelewska, M., Mąka, L., Ścieżyńska, H., Pawłowska, K., Postupolski, J., et al. (2016). Antimicrobial resistance profiles of Listeria monocytogenes isolated from ready-to-eat products in Poland in 2007-2011. Food Control, 59, 7–11. https://doi. org/10.1016/j.foodcont.2015.05.011. Mainar, M. S., Xhaferi, R., Samapundo, S., Simba, R., Frank, D., & Frédéric, L. (2016). Opportunities and limitations for the production of safe fermented meats without nitrate and nitrite using an antibacterial Staphylococcus sciuri starter culture. Food Control, 69, 267–274. https://doi.org/10.1016/j.foodcont.2016.04.056. Martinho, G., Balaia, N., & Pires, A. (2017). The Portuguese plastic carrier bag tax: The effects on consumers' behavior. Waste Management, 61, 3–12. https://doi.org/10.
163