Antonie van Leeuwenhoek (2014) 105:413–421 DOI 10.1007/s10482-013-0073-4
SHORT COMMUNICATION
Multilocus sequence typing and antimicrobial resistance in Enterococcus faecium isolates from fresh produce Ma Jose´ Grande Burgos • Ma Carmen Lo´pez Aguayo Rube´n Pe´rez Pulido • Antonio Ga´lvez • Rosario Lucas Lo´pez
•
Received: 28 July 2013 / Accepted: 7 November 2013 / Published online: 19 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The purpose of the present study was to determine the relatedness of Enterococcus faecium isolates from fresh produce to E. faecium strains from other sources by using multi-locus sequence typing (MLST) and to determine the antimicrobial resistance of the isolates. MLST analysis of 22 E. faecium isolates from fresh produce revealed 7 different sequence types (ST 22, ST 26, ST 43, ST 46, ST 55, ST 94 and ST 296). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). All isolates were sensitive to vancomycin and to imipenem, and only one was resistant to ampicillin (MIC 32 mg/l). However, all were resistant to cefotaxime and ceftazidine. E. faecium isolates from fresh produce were inhibited by quaternary compounds (benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide), biguanides (chlorhexidine), polyguanides [poly-(hexamethylene guanidinium) hydrochloride], bisphenols (triclosan, hexachlorophene) and biocidal solutions of P3 oxonia and P3 topax 66. Didecyldimethylammonium bromide and triclosan were the least effective biocides in growth inhibition, while hexadecylpyridinium chloride was the most effective. Results from MLST typing and
M. J. G. Burgos (&) M. C. L. Aguayo R. P. Pulido A. Ga´lvez R. L. Lo´pez ´ rea de Microbiologı´a, Departamento de Ciencias de la A Salud, Facultad de Ciencias Experimentales, Universidad de Jae´n, Campus Las Lagunillas s/n, 23071 Jae´n, Spain e-mail: [email protected]
antibiotic resistance suggest that the studied E. faecium isolates from fresh produce are not related to the clinically-relevant clonal complex CC17. Keywords MLST
Enterococci Fresh produce
Introduction Enterococci are widely distributed in nature as commensal or saprophytic bacteria, being frequently found in foods (Giraffa 2002; Koluman et al. 2009; Ogier and Serror 2008). At the same time, they rank among leading causes of hospital acquired infections of the bloodstream, urinary tract, surgical wounds and other sites (Arias and Murray 2012; Hidron et al. 2008). Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumably due to expansion of a human-associated polyclonal multilocus sequence typing (MLST) subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin, vancomycin and quinolone resistance) and virulence traits (Freitas et al. 2010). Recent studies indicates that E. faecium CC17 clinical isolates identified to date group in a subcluster composed of different clones that have evolved independently, although they still might be genetically linked (Naser et al. 2005; Vankerckhoven et al. 2008; Willems and
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van Schaik 2009). Half of the sequence types (STs) within this CC17 polyclonal subpopulation have also been identified from samples obtained from healthy humans, swine, poultry, and pets (Willems and van Schaik 2009). Hospital E. faecium isolates could be divided into three lineages originating from STs 17, 18, and 78 (Willems et al. 2012). It was proposed that these genetic lineages originated by the selective pressure of hospitals and have become gradually isolated from other populations (Willems et al. 2012). This would also suggest evolution from a commensal bacterium to a hospital adapted pathogen (Gilmore et al. 2013; Willems and van Schaik 2009). The possible relatedness of enterococci from foods and those from clinical settings is a matter of concern. Most studies on MLST typing have focused on clinical isolates along with some isolates from animals and from foods of animal origin. However, information on MLST types from other environments such as vegetable foods is scarce or unavailable. Previous studies have reported the detection of enterococci on plants (Mu¨ller et al. 2001; Mundt 1961, 1963; Mundt et al. 1962; Ott et al. 2001; Ulrich and Mu¨ller 1998), wild flowers (Sa´nchez Valenzuela et al. 2012), fermented vegetables (Pe´rez-Pulido et al. 2006) as well as in fresh produce (Abriouel et al. 2008; Campos et al. 2013; Johnston and Jaykus 2004; Johnston et al. 2005; McGowan et al. 2006; Micallef et al. 2013). MLST typing of enterococci from plant rhizospheres and of Enterococcus faecalis from vegetable foods revealed new sequence types (Klibi et al. 2012; Grande Burgos et al. 2009). Regardless of their sources of isolation, enterococci may harbour antimicrobial resistance traits (Peters et al. 2003; Rice et al. 2009; Hollenbeck and Rice 2012; Gilmore et al. 2013). Antimicrobial resistant strains may be transmitted to humans through the food chain increasing the risk for opportunistic infections that may be difficult to treat by conventional antimicrobial therapy. Since data on possible relatedness of E. faecium isolates from vegetable foods to human or animal isolates is scarce, the main purpose of the present study was to determine the sequence type of 22 E. faecium isolates from fresh produce and compare them with existing STs and CCs in the MLST database. Isolates were further investigated regarding sensitivity to antibiotics and biocides in order to gain insights into their antimicrobial resistance levels.
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Materials and methods Bacterial isolates and cultivation conditions A collection of 22 E. faecium isolates from fresh produce (Abriouel et al. 2008) were used for the present study (Table 1). Isolates were obtained from Table 1 MLST type and antibiotic resistance in E. faecium isolates from fresh produce Isolate
Source
MLST type
Antibiotic resistance
V2C1
Green olives
296
CAZ, CTX, ERY, TET, NA, FUR
V11C1
Green olives
296
CAZ, CTX, RFA
V12C1
Beet
296
CAZ, CTX, ERY, RFA, CIP, NA, NET
V14C1
Beet
296
NA
V39C4
Packed Mediterranean salad
296
AMP, CAZ, CTX, ERY, TET
V50C1
Lettuce
296
CAZ, CTX, ERY, RFA, CIP
V55C2
Lettuce
296
CAZ, CTX, ERY, RFA, CIP, NET
V59C1
Potato
296
CAZ, CTX, RFA
V73C1
Tomato
296
CAZ, CTX, RFA
V18C2
Alfalfa sprouts
94
CAZ, CTX, ERY
V35C1
Dates
94
CAZ, CTX, ERY, RFA
V36C1
Endives
94
CAZ, CTX, ERY, RFA, CIP, NA, NET
V37C1
Packed spring salad
94
CAZ, CTX, RFA, CIP
V70C1
Soybean sprouts
94
CAZ, CTX, ERY, RFA, TET, CIP
V71C3
Soybean sprouts
94
CAZ, CTX, ERY, RFA, CIP
V51C1
Lettuce
22
CAZ, CTX, RFA, TET, CIP, NA, NET, TMP/ STX
V26C1
Brocoli
26
CAZ, CTX, TET, CIP
V47C2
Strawberries
43
CAZ, CTX, TET, CIP, NA,
V16C1
Artichoke
46
CAZ, CTX, CIP
V17C1
Alfalfa sprouts
46
CAZ, CTX
V3C2
Green olives
55
CAZ, CTX, ERY, TET
V57C2
Celery
55
CAZ, CTX, ERY, TET
AMP ampicillin, CAZ ceftazidine, CTX cefotaxime, ERY erythromycin, RFA rifampin, TET tetracycline, CIP ciprofloxacin, NA nalidixic acid, NET netilmicin, TMP/STX trimethoprim/sulfamethoxazol, FUR nitrofurantoin
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food samples purchased in 15 different supermarkets located in the South of Spain. Each sample of the same food type was from a different supermarket. Isolates were cultivated routinely on brain heart infusion (BHI) broth (Scharlab, Barcelona, Spain) at 37 °C. Isolates were stored at -80 °C in BHI containing 20 % glycerol. MLST analysis The isolates were analyzed by MLST for E. faecium (http://efaecium.mlst.net; Homan et al. 2002) by using primer sequences available at http://efaecium.mlst.net/ misc/info.asp. The allelic profiles of isolates were obtained by sequencing internal fragments of seven housekeeping genes: adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine: D-alanine ligase), gdh (glucose-6-phosphate dehydrogenase), gyd (glyceraldehyde-3-phosphate dehydrogenase), purK (phosphoribosylaminoimidazol carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter). PCR conditions for all amplification reactions were as follows: initial denaturation at 94 °C for 3 min; 35 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; and extension at 72 °C for 5 min. Reactions were performed in 50 ll volumes with buffers and Taq polymerase. PCR products were purified with ExoSAP-IT (USB Europe GmbH) and sequenced with the respective primers in a CEQ 2000 XL DNA Analysis System (Beckman Coulter, CA). For every isolate, each gene was amplified at least two times and sequenced with the specific forward or reverse primer at least three times. The obtained sequences were submitted to the E. faecium MLST database (http://www. mlst.net) for assignment of alleles at each locus and a sequence type. Cluster analysis of the data was performed using the MLST database and the e-BURST algorithm. Sequence types of isolates are defined by the allelic profile at these seven loci, with each unique combination of alleles assigned a distinct sequence type number and the sequence types (STs) of all isolates were determined. Determination of antibiotic resistance Antibiotic resistance determination was performed by the disk diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI 2011) on Mueller–Hinton agar (Scharlab, Barcelona, Spain),
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choosing antibiotics that are widely used or frequently included in studies of antimicrobial resistance in enterococci. Ampicillin (AMP, 30 lg), ceftazidine (CAZ, 30 lg), cefotaxime (CTX, 30 lg), imipenem (IPM, 10 lg), erythromycin (ERY, 15 lg), tetracycline (TET, 30 lg), ciprofloxacin (CIP, 5 lg), nalidixic acid (NA, 30 lg), netilmicin (NET, 30 lg), trimethoprim/ sulfamethoxazole (TMP/STX, 25/75 lg), nitrofurantoin (FUR, 300 lg) were from Biome´rieux (Madrid, Spain). Vancomycin (VAN, 30 lg), chloramphenicol (CMP, 30 lg) and rifampin (RIF, 5 lg) were from BBL (Madrid, Spain). The levels of antibiotic resistance for ampicillin (GlaxoSmithKline, Madrid), ceftazidime and cefotaxime (borth from Normon, Madrid) were also determined by broth microdilution tests on Mueller– Hinton broth following CLSI guidelines and using antibiotic concentrations of 2, 4, 6, 8, 12, 16, 32 and 64 mg/l. Assays were performed in 96-well microtiter plates. Optical density at 595 nm was recorded at time 0 and 24 h of incubation at 37 °C with an iMark Microplate Reader (BioRad, Madrid). Determination of biocide tolerance Benzalkonium chloride (BC), cetrimide (CT), hexadecylpyridinium chloride (HDP), didecyldimethylammonium bromide (AB), chlorhexidine (CH), triclosan (TC) and hexachlorophene were from Sigma-Aldrich (Madrid, Spain). Benzalkonium chloride commercial solution contained 50 % (w/v) of the active compound. Triclosan and hexachlorophene were dissolved (10 % w/v) in 96 % ethanol. All other biocides were dissolved aseptically in sterile distilled water at final concentrations of 5 % (CH and HDP) or 10 % (CT), and stored at 4 °C for B7 days. Poly-(hexamethylene guanidinium) hydrochloride (PHMG) solution (containing 7.8 % of PHMG, by weight) was a kind gift of Oy Soft Protector Ltd (Espoo, Finland). The commercial sanitizers P3 oxonia (25–35 % hydrogen peroxide, 0.83–2.5 N acetic acid and 0.26–0.66 N peracetic acid) and P3 topax 66 (2–5 % sodium hydroxide, 2–5 % sodium hypochlorite and 2–5 % alkylamine) were obtained from ECOLAB (Barcelona, Spain). Minimal inhibitory concentrations (MICs) to biocides were determined by the microbroth dilution method on 96-well bottom microtiter plates (Becton–Dickinson Labware, Franklin Lakes, NJ). Briefly, serial dilutions of biocides were incubated with bacterial suspensions adjusted to 5 9 105 CFU/ml in Mueller–Hinton Broth
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(Scharlab). Growth controls (inoculated growth medium without antimicrobials) and sterility controls (non-inoculated medium supplemented with antimicrobials) were included. Microtiter plates were incubated at 37 °C and readings were performed after 24 h of incubation by optical density (OD 595 nm) determination in an iMark Microplate Reader. MICs were determined as the minimum biocide concentration that achieved complete growth inhibition.
Results and discussion The 22 E. faecium isolates from fresh produce investigated comprised 7 different MLST types (STs 22, 26, 43, 46, 55, 94 and 296; Table 1; Fig. 1). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). Other STs (46 and 55) were represented by two isolates (9.1 %) or just one isolate (STs 22, 26,
ST43
ST46 ST 55
ST 22
78
CC17
Fig. 1 Population snapshot of E. faecium. The entire E. faecium MLST database is displayed as a single eBURST diagram. STs (nodes in the diagram) are labeled and STs differing in only one allele (i.e. single locus variants) are connected by arcs. Colors indicate predicted primary (blue) and
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43). The STs most represented in E. faecium from fresh produce only have a few representatives in the MLST database, most of them from clinical sources. For example, ST 296 includes two strains from hospitalized patients, one from a cat belly and one probiotic strain. The closest STs with six alleles in common (345, 694 and 695) include strains from blood of a hospitalized patient and from non-hospitalized persons. ST 94 is closely related to 12 additional STs and includes strains that were isolated from faeces of healthy people and from hospitalized patients. ST 22 is highly represented in the MLST database and includes strains from hospitalized patients (four of them being vancomycin-resistant), two vancomycin-resistant strains from cheese and two strains from pig faeces. ST 22 is strongly related (6 common alleles) to 24 other STs. ST 26 includes strains from chicken and turkey (VanA) faeces, hospitalized patients and a non-specified source.
ST 26
ST 94 ST 296
secondary (yellow) founders. The areas of each of the circles indicate the prevalence of the ST in the input data. The STs found in this study and the STs 17, 18 and 78 belonging to clonal complex 17 (CC17) are marked by arrows
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ST43 includes strains from calf faeces and from meat. Its two closest STs (172, 187) include VanA isolates from calf faeces. ST 46 includes one strain from a hospitalized patient. Its closest STs (69, 161 and 704) include isolates from river water and faeces from nonhospitalized persons. There are few studies on MLST typing of enterococci from vegetable foods. In one study on E. faecalis, Grande Burgos et al. (2009) reported that most isolates belonged to ST 168. However, one isolate belonged to ST 9 of the previously-described clonal complex (CC) 9 which is frequently associated with hospital environments. In a recent study, E. faecium isolates from the rhizosphere were assigned to STs 21, 606, 613, 614 and 615, demonstrating new genetic lineages among rhizosphere isolates (Klibi et al. 2012). Since many of the isolates included in the MLST database originate from clinical sources, it is important to also investigate isolates from other sources such as the environment or fresh produce in order to achieve a more complete picture of the population diversity and relatedness of E. faecium isolates. The antibiotic resistance of isolates is shown in Table 1. None of isolates was resistant to vancomycin. All isolates were sensitive to imipenem and only one isolate (V39C4 from ST 296) was resistant to ampicillin (MIC 32 mg/l). E. faecium has a high capacity to acquire high levels of ampicillin resistance under selective pressure of this antibiotic (Rice et al. 2001). Ampicillin resistance is now recognized as a marker for strains belonging to CC17 (Top et al. 2008). All isolates were resistant to cefotaxime and ceftazidine. MICs for cefotaxime were[64 mg/l for most isolates, except V11C1 (32 mg/l) or V36C1 (64 mg/l). MICs for ceftazidine ranged from 16 mg/l (isolate V11C1) to 32 mg/l (V3C2, V50C1), 64 mg/l (V38C1) or [64 mg/l (rest of isolates). In enterococci, betalactam resistance is mediated by Pbp5 in cooperation with class A PBPs (Rice et al. 2001). However, resistance to ampicillin and cephalosporins can be dissociated by the deletion of the class A pbp genes, pbpF and pbpA, indicating a complex mechanism of beta-lactam resistance in this bacterium (Rice et al. 2009). Enterococci are intrinsically resistant to cephalosporins (Murray 1990) and cephalosporin administration may select for enterococcal infections (Linden and Miller 1999). Netilmicin resistance was detected in four isolates in the present study but
417
according to previous data (Abriouel et al. 2008), all isolates were sensitive to gentamicin. Aminoglycosides such as netilmicin or gentamicin are recommended for the treatment of E. faecalis infections, especially in severe and bacteremic infection (Dube´ et al. 2012). In a previous study, we reported that erythromycin and rifampicin were the most frequent antibiotic resistance traits in E. faecium from vegetable foods, followed by ciprofloxacin and tetracycline (Abriouel et al. 2008). These results were also confirmed in the present study. The use of antibiotics such as erythromycin or tetracycline in animal husbandry and agriculture, and the spread of resistant strains through polluted irrigation waters, animal faeces or insects seem to be contributing factors for the presence of antibioticresistant enterococci in the environment (Hollenbeck and Rice 2012; Sa´nchez Valenzuela et al. 2012; Furtula et al. 2013; Micallef et al. 2013). Enterococci carrying antibiotic resistance traits have been frequently found in tomatoes, as well as in the irrigation waters and soil (Micallef et al. 2013). Ground distance and type of farm (small vs. large farms) influenced the incidence of enterococci in tomatoes. It has been suggested that, unlike large-scale farms that fertilized exclusively with chemical nutrients, small-scale farms occasionally used poultry litter for fertilization. The current recommended composting times for poultry litter may not be long enough to eliminate all enterococci (Graham et al. 2009). Enterococci from our study were isolated from a variety of vegetable foods bought at 15 different supermarkets and obtained by different cultivation practices. Most of them grow a short distance from the ground (such as lettuce, broccoli, endives, celery or asparagus), deep in the ground (such as beet or potato) or in hydroponic cultures (such as sprouts). Therefore, contamination of many of these foods by enterococci from environmental sources seems very likely. Many of these foods are sold without being washed (like for example whole lettuce or endives), while others may be washed and processed, such as the ready to eat salads. Conventional chlorine washing steps may be insufficient to inactivate enterococci in salads, as shown in a recent study (Campos et al. 2013). Chlorine disinfection is widely used on fresh produce. In addition, many other biocidal substances are used by the food industry for cleaning and disinfection practices (McDonnel and Russell 1999; Cerf et al.
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active biocide among quaternary ammonium compounds, requiring 500 mg/l or even 750 mg/l for two isolates. Previous studies have reported MICs for BC of 2–4 mg/l in E. faecium isolates from dust in breeding pigs facilities (Braga et al. 2013), or between 2 and 16 mg/l in E. faecium isolates from livestock (Aarestrup and Hasman 2004). Sidhu et al. (2002) proposed that Enterococcus sp. isolates with MICs for BC \30 mg/l should be considered sensitive to this biocide, while 30–50 mg/l was considered as low-level resistance and [50 mg/l was considered as high-level resistance. In another study, between 92.5 and 100 % of enterococci from foods, animals or clinical sources were inhibited by 25 mg/l BC, and most isolates also were inhibited by 25 mg/l CT or 2.5 mg/l HDP (Valenzuela et al. 2013). All isolates from the present study were inhibited at 75 mg/l of the biguanide CH, while MICs for the
2010). Food processing facilities and utensils such as transport containers, boxes, conveyor belts, cutting and peeling machines, floor drains or storage rooms where organic matter accumulates frequently become sources of bacterial contamination and need to be disinfected periodically. These may be potential sources of enterococci exposed to different types of biocides that may then enter the food chain. The results obtained in the present study on biocide tolerance of E. faecium isolates from fresh produce are shown in Table 2. Among the quaternary ammonium compounds, the most active biocide was HDP, which inhibited most isolates at 1 mg/l. For BC, 40.9 % of isolates were inhibited at 12.5 mg/l, while others required 25 or 37.5 mg/l, and one isolate was inhibited only at 50 mg/l (Table 2). Most isolates required 75 mg/l CT for inhibition but some were also inhibited at 25 mg/l. AB was the least
Table 2 Biocide tolerance in E. faecium isolates from fresh produce Biocide concentration (mg/l)
Biocide MIC distribution (%) BC
CT
HDP
1
95.46
2
4.54
AB
CH
PHMG
TC
5
CF
OX
TOP
95.45
100
22.73
8
27.27
10 12.5
13.63 40.91
20
40.91
25
22.73
37.5
31.81
50
4.55
31.82 4.54
63.64
60 75
31.82 63.64
100
100 500
90.91
27.28
750 0.25a
9.09
72.72
0.5a 0.75a
4.55
Minimal inhibitory concentrations and their corresponding percentages of isolates are shown Ranges of biocide concentrations recommended by manufacturers: BC, 0.05–0.2 %; CT, 0.1–0.5 %; HDP, up to 0.8 %; AB, 0.01–0.1 %; CH, 0.01–4.0 %; PHMG, 0.1–0.2 %; TC, 0.3–2 %; CF, up to 0.1 %; OX and TOP, 0.05 to 5 % BC benzalkonium chloride, CT cetrimide, HDP hexadecylpyridinium chloride, AB didecyldimethylammonium bromide, CH chlorhexidine, PHMG poly-(hexamethylen guanidinium) hydrochloride, TC triclosan, CF hexachlorophene OX P3 oxonia commercial solution, TOP P3 topax 66 commercial solution a
Biocide concentration is expressed as % of commercial solution (v/v)
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polyguanide PHMG ranged from 8 to 60 mg/l (Table 2). Reported MICs for CH from other studies on enterococci were of 2 mg/l or lower (Beier et al. 2008), 2–4 mg/l (Braga et al. 2013), 4–6 mg/l (Suller and Russell 1999) or even up to 2,500 mg/l in clinical isolates (Valenzuela et al. 2013). Formulations containing biocides such as BC, CH and PHMG are used for sanitizing the surfaces of utensils and instruments in the food industry at concentrations of 0.05–0.2, 0.01–0.02, and 0.1–0.2 % of the active ingredient, respectively (Ueda and Kuwabara 2007). CH is also frequently used in personal care and hand hygiene products at concentrations of 2–4 % (Kampf and Kramer 2004). HDP was approved in 2004 by the USA Food and Drug Administration for decontaminating raw poultry at a working concentration not exceeding 0.8 % by weight (Food and Drug Administration 2004). Among the bisphenols tested, TC required concentrations of 500–750 mg/l for inhibition, while CF was active at lower concentrations but it had a broader range of MICs, from 5 to 50 mg/l (Table 2). Other studies on enterococci have reported MICs for TC from 3 to 4 mg/l (Suller and Russell 1999), 8 mg/l (Beier et al. 2008) or even higher than 250 mg/l (Valenzuela et al. 2013). TC is now incorporated in a wide range of products, such as dishcloths, food boxes, toothbrushes, washing-up liquid and hand-washing gels, plastics, chopping boards, chopsticks, pizza cutters, food storage containers, garbage bags, or kitty litter, among others, at recommended concentrations that may range from 0.3 to 1 % (Yazdankhah et al. 2006) or even 1–2 % (Kampf and Kramer 2004). The commercial solutions P3 oxonia and P3 topax 66 were active on E. faecium isolates at final concentrations of 0.25–0.75 % by volume (Table 2), which is in the range of their recommended use concentrations of 0.05–5 % (Blakistone et al. 1999). There is also a concern that exposure to biocides and biocide tolerance may co-select for antibiotic resistance (Ortega-Morente et al. 2013). However, previous studies have shown that antibiotic-resistant enterococci are sensitive or moderately resistant to biocides such as BC, TC or CH (Suller and Russell 1999; Fraise 2002; Aarestrup and Hasman 2004; Beier et al. 2008). In the present study, those isolates showing greater numbers of antibiotic resistance traits had lowest MICs for biocides, as in the case of isolate V12C1 (which had lowest MICs for BC, CT, CF, AB, TC and HDP) and isolate V51C1 (with lowest MICs for BC, CT and HDP). These results
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may be an indication that antibiotic resistance and biocide tolerance are not related. Another concern about enterococci is the presence of virulence factors (Gilmore et al. 2013). According to our previous study, the incidence of virulence factors among E. faecium isolates from vegetable foods was much lower compared to clinical isolates, although 45.5 % of isolates carried the gelatinase gene gelE (Abriouel et al. 2008). None of isolates showed betahaemolytic activity, and none carried the complete set of cytolysin genes. While 54.5 % of isolates carried cylB, only 4.5 % carried cylA or cylM. Other virulence genes, such as aggregation substance (agg) and enterococcal surface protein (esp) were detected at low frequencies (18.2 and 27.3 %) compared to clinical isolates, while other genetic determinants frequently found in clinical isolates such as the collagen adhesin (ace) and the E. faecium endocarditis gene (efafm) were not detected (Abriouel et al. 2008). These previous results are in agreement with the observed lack of resistance to antibiotics such as vancomycin or ampicillin typically found in clinical isolates and also with the obtained MLST data, suggesting that the studied isolates belong to subpopulations not related to nosocomial strains. In conclusion, results from the present study indicate that the E. faecium isolates from the fresh produce analysed are not related to the clinically relevant clonal complex CC17. Some STs (especially ST 296 but also ST 94) seem to be more common in different types of fresh produce. Antimicrobial resistance seems to be widely disseminated among the studied enterococci from fresh produce, but their biocide tolerance levels fall below the recommended use concentrations of biocides. Acknowledgments This work was supported by research project P08-AGR-4295 (Junta de Andalucı´a CICE, FEDER) and University of Jae´n (Plan Propio). We also acknowledge the Campus de Excelencia Agroalimentario CeiA3. Conflict of interest The authors declare that they have no conflict of interest.
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Antonie van Leeuwenhoek (2014) 105:413–421 Graham JP, Evans SL, Price LB, Silbergeld EK (2009) Fate of antimicrobial resistant enterococci and staphylococci and resistance determinants in stored poultry litter. Environ Res 109:682–689 Grande Burgos MJ, Lucas Lo´pez R, Abriouel H, Ben Omar N, Galvez A (2009) Multilocus sequence typing of Enterococcus faecalis from vegetable foods reveals two new sequence types. Foodborne Pathog Dis 6:321–327 Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK (2008) National Healthcare Safety Network Team and participating National Healthcare Safety Network Facilities: NHSN annual update: antimicrobialresistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 29:996–1011 Hollenbeck BL, Rice LB (2012) Intrinsic and acquired resistance mechanisms in Enterococcus. Virulence 3:421–433 Homan WL, Tribe D, Poznanski S, Li M, Hogg G, Spalburg E, Van Embden JD, Willems RJ (2002) Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol 40:1963–1971 Johnston LM, Jaykus LA (2004) Antimicrobial resistance of Enterococcus species isolated from produce. Appl Environ Microbiol 70:3133–3137 Johnston LM, Jaykus LA, Moll D, Martinez MC, Anciso J, Mora B, Moe CL (2005) A field study of the microbiological quality of fresh produce. J Food Prot 68:1840–1847 Kampf G, Kramer A (2004) Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs. Clin Microbiol Rev 17:863–893 Klibi N, Ben Slimen N, Fhoula I, Lo´pez M, Ben Slama K, Daffonchio D, Boudabous A, Torres A, Ouzari H (2012) Genotypic diversity, antibiotic resistance and bacteriocin production of enterococci isolated from rhizospheres. Microbes Environ 27:533–537 Koluman A, Akan LS, Pınar C¸akirog˘lu F (2009) Occurrence and antimicrobial resistance of enterococci in retail foods. Food Control 20:281–283 Linden PK, Miller CB (1999) Vancomycin-resistant enterococci: the clinical effect of a common nosocomial pathogen. Diagn Microbiol Infect Dis 33:113–120 McDonnel G, Russell AD (1999) Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 12:147–179 McGowan LL, Jackson CR, Barrett JB, Hiott LM, Fedorka-Cray PJ (2006) Prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats. J Food Prot 69:2976–2982 Micallef SA, Rosenberg Goldstein RE, George A, Ewing L, Tall BD, Boyer MS, Joseph SW, Sapkota AR (2013) Diversity, distribution and antibiotic resistance of Enterococcus spp. recovered from tomatoes, leaves, water and soil on U.S. Mid-Atlantic farms. Food Microbiol 36:465–474 Mu¨ller T, Ulrich A, Ott EM, Mu¨ller M (2001) Identification of plant-associated enterococci. J Appl Microbiol 91:268–278 Mundt JO (1961) Occurrence of enterococci on plants: bud, blossom, and soil studies. Appl Microbiol 9:541–544 Mundt JO (1963) Occurrence of enterococci on plants in a wild environment. Appl Microbiol 11:141–144
Antonie van Leeuwenhoek (2014) 105:413–421 Mundt JO, Coggins JH, Johnson LF (1962) Growth of Streptococcus faecalis var. liquefaciens on plants. Appl Microbiol 10:552–555 Murray BE (1990) The life and times of the Enterococcus. Clin Microbiol Rev 3:46–65 Naser S, Thompson FL, Hoste B, Gevers D, Vandemeulebroecke K, Cleenwerck I, Thompson CC, Vancanneyt M, Swings J (2005) Phylogeny and identification of enterococci by atpA gene sequence analysis. J Clin Microbiol 43:2224–2230 Ogier JC, Serror P (2008) Safety assessment of dairy microorganisms: the Enterococcus genus. Int J Food Microbiol 126:291–301 Ortega-Morente E, Ferna´ndez-Fuentes MA, Grande-Burgos MJ, Abriouel H, Pe´rez-Pulido R, Ga´lvez A (2013) Biocide tolerance in bacteria. Int J Food Microbiol 162:13–25 Ott EM, Muller T, Muller M, Franz CMAP, Ulrich A, Gabel M, Seyfarth L (2001) Population dynamics and antagonistic potential of enterococci colonizing the phyllosphere of grasses. J Appl Microbiol 91:54–66 Pe´rez-Pulido R, Abriouel H, Ben Omar N, Lucas R, Martı´nezCan˜amero M, Ga´lvez A (2006) Safety and potential risks of enterococci isolated from traditional fermented capers. Food Chem Toxicol 44:2070–2077 Peters J, Mac K, Wichmann-Schauer H, Klein G, Ellerbroek L (2003) Species distribution and antibiotic resistance patterns of enterococci isolated from food of animal origin in Germany. Int J Food Microbiol 88:311–314 Rice LB, Carias LL, Hutton-Thomas R, Sifaoui F, Gutmann L, Rudin SD (2001) Penicillin-binding protein 5 and expression of ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 45:1480–1486 Rice LB, Carias LL, Rudin S, Hutton R, Marshall S, Hassan M, Josseaume N, Dubost L, Marie A, Arthur M (2009) Role of class A penicillin-binding proteins in the expression of blactam resistance in Enterococcus faecium. J Bacteriol 191:3649–3656 Sa´nchez Valenzuela A, Benomar N, Abriouel H, Pe´rez Pulido R, Ga´lvez A (2012) Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers. Antonie Van Leeuwenhoek 101:701–711
421 Sidhu MS, Sørum H, Holck A (2002) Resistance to quaternary ammonium compounds in food-related bacteria. Microb Drug Resist 8:393–399 Suller MT, Russell AD (1999) Antibiotic and biocide resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. J Hosp Infect 43:281–291 Top J, Willems R, Bonten M (2008) Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen. FEMS Immunol Med Microbiol 52:297–308 Ueda S, Kuwabara Y (2007) Susceptibility of biofilm Escherichia coli, Salmonella enteritidis and Staphylococcus aureus to detergents and sanitizers. Biocontrol Sci 12:149–153 Ulrich A, Mu¨ller T (1998) Heterogeneity of plant-associated streptococci as characterised by phenotypic features and restriction analysis of PCR-amplified 16S rDNA. J Appl Microbiol 84:293–303 Valenzuela AS, Benomar N, Abriouel H, Can˜amero MM, Lo´pez RL, Ga´lvez A (2013) Biocide and copper tolerance in enterococci from different sources. J Food Prot 76:1806–1809 Vankerckhoven V, Huys G, Vancanneyt M, Snauwaert C, Swings J, Klare I, Witte W, Van Autgaerden T, Chapelle S, Lammens C, Goossens H (2008) Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates. Appl Environ Microbiol 74:4247–4255 Willems RJL, van Schaik W (2009) Transition of Enterococcus faecium from commensal organism to nosocomial pathogen. Future Microbiol 4:1125–1135 Willems RJL, Top J, van Schaik W, Leavis H, Bonten M, Sire´n J, Hanage WP, Corander J (2012) Restricted gene flow among hospital subpopulations of Enterococcus faecium. MBio 3:e00151–e00212 Yazdankhah SP, Scheie AA, Høiby EA, Lunestad B-T, Heir E, Fotland TØ, Naterstad K, Kruse H (2006) Triclosan and antimicrobial resistance in bacteria: an overview. Microb Drug Resist 12:83–90
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SHORT COMMUNICATION
Multilocus sequence typing and antimicrobial resistance in Enterococcus faecium isolates from fresh produce Ma Jose´ Grande Burgos • Ma Carmen Lo´pez Aguayo Rube´n Pe´rez Pulido • Antonio Ga´lvez • Rosario Lucas Lo´pez
•
Received: 28 July 2013 / Accepted: 7 November 2013 / Published online: 19 November 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract The purpose of the present study was to determine the relatedness of Enterococcus faecium isolates from fresh produce to E. faecium strains from other sources by using multi-locus sequence typing (MLST) and to determine the antimicrobial resistance of the isolates. MLST analysis of 22 E. faecium isolates from fresh produce revealed 7 different sequence types (ST 22, ST 26, ST 43, ST 46, ST 55, ST 94 and ST 296). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). All isolates were sensitive to vancomycin and to imipenem, and only one was resistant to ampicillin (MIC 32 mg/l). However, all were resistant to cefotaxime and ceftazidine. E. faecium isolates from fresh produce were inhibited by quaternary compounds (benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide), biguanides (chlorhexidine), polyguanides [poly-(hexamethylene guanidinium) hydrochloride], bisphenols (triclosan, hexachlorophene) and biocidal solutions of P3 oxonia and P3 topax 66. Didecyldimethylammonium bromide and triclosan were the least effective biocides in growth inhibition, while hexadecylpyridinium chloride was the most effective. Results from MLST typing and
M. J. G. Burgos (&) M. C. L. Aguayo R. P. Pulido A. Ga´lvez R. L. Lo´pez ´ rea de Microbiologı´a, Departamento de Ciencias de la A Salud, Facultad de Ciencias Experimentales, Universidad de Jae´n, Campus Las Lagunillas s/n, 23071 Jae´n, Spain e-mail: [email protected]
antibiotic resistance suggest that the studied E. faecium isolates from fresh produce are not related to the clinically-relevant clonal complex CC17. Keywords MLST
Enterococci Fresh produce
Introduction Enterococci are widely distributed in nature as commensal or saprophytic bacteria, being frequently found in foods (Giraffa 2002; Koluman et al. 2009; Ogier and Serror 2008). At the same time, they rank among leading causes of hospital acquired infections of the bloodstream, urinary tract, surgical wounds and other sites (Arias and Murray 2012; Hidron et al. 2008). Enterococcus faecium has increasingly been reported as a nosocomial pathogen since the early 1990s, presumably due to expansion of a human-associated polyclonal multilocus sequence typing (MLST) subcluster known as clonal complex 17 (CC17) that has progressively acquired different antibiotic resistance (ampicillin, vancomycin and quinolone resistance) and virulence traits (Freitas et al. 2010). Recent studies indicates that E. faecium CC17 clinical isolates identified to date group in a subcluster composed of different clones that have evolved independently, although they still might be genetically linked (Naser et al. 2005; Vankerckhoven et al. 2008; Willems and
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van Schaik 2009). Half of the sequence types (STs) within this CC17 polyclonal subpopulation have also been identified from samples obtained from healthy humans, swine, poultry, and pets (Willems and van Schaik 2009). Hospital E. faecium isolates could be divided into three lineages originating from STs 17, 18, and 78 (Willems et al. 2012). It was proposed that these genetic lineages originated by the selective pressure of hospitals and have become gradually isolated from other populations (Willems et al. 2012). This would also suggest evolution from a commensal bacterium to a hospital adapted pathogen (Gilmore et al. 2013; Willems and van Schaik 2009). The possible relatedness of enterococci from foods and those from clinical settings is a matter of concern. Most studies on MLST typing have focused on clinical isolates along with some isolates from animals and from foods of animal origin. However, information on MLST types from other environments such as vegetable foods is scarce or unavailable. Previous studies have reported the detection of enterococci on plants (Mu¨ller et al. 2001; Mundt 1961, 1963; Mundt et al. 1962; Ott et al. 2001; Ulrich and Mu¨ller 1998), wild flowers (Sa´nchez Valenzuela et al. 2012), fermented vegetables (Pe´rez-Pulido et al. 2006) as well as in fresh produce (Abriouel et al. 2008; Campos et al. 2013; Johnston and Jaykus 2004; Johnston et al. 2005; McGowan et al. 2006; Micallef et al. 2013). MLST typing of enterococci from plant rhizospheres and of Enterococcus faecalis from vegetable foods revealed new sequence types (Klibi et al. 2012; Grande Burgos et al. 2009). Regardless of their sources of isolation, enterococci may harbour antimicrobial resistance traits (Peters et al. 2003; Rice et al. 2009; Hollenbeck and Rice 2012; Gilmore et al. 2013). Antimicrobial resistant strains may be transmitted to humans through the food chain increasing the risk for opportunistic infections that may be difficult to treat by conventional antimicrobial therapy. Since data on possible relatedness of E. faecium isolates from vegetable foods to human or animal isolates is scarce, the main purpose of the present study was to determine the sequence type of 22 E. faecium isolates from fresh produce and compare them with existing STs and CCs in the MLST database. Isolates were further investigated regarding sensitivity to antibiotics and biocides in order to gain insights into their antimicrobial resistance levels.
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Materials and methods Bacterial isolates and cultivation conditions A collection of 22 E. faecium isolates from fresh produce (Abriouel et al. 2008) were used for the present study (Table 1). Isolates were obtained from Table 1 MLST type and antibiotic resistance in E. faecium isolates from fresh produce Isolate
Source
MLST type
Antibiotic resistance
V2C1
Green olives
296
CAZ, CTX, ERY, TET, NA, FUR
V11C1
Green olives
296
CAZ, CTX, RFA
V12C1
Beet
296
CAZ, CTX, ERY, RFA, CIP, NA, NET
V14C1
Beet
296
NA
V39C4
Packed Mediterranean salad
296
AMP, CAZ, CTX, ERY, TET
V50C1
Lettuce
296
CAZ, CTX, ERY, RFA, CIP
V55C2
Lettuce
296
CAZ, CTX, ERY, RFA, CIP, NET
V59C1
Potato
296
CAZ, CTX, RFA
V73C1
Tomato
296
CAZ, CTX, RFA
V18C2
Alfalfa sprouts
94
CAZ, CTX, ERY
V35C1
Dates
94
CAZ, CTX, ERY, RFA
V36C1
Endives
94
CAZ, CTX, ERY, RFA, CIP, NA, NET
V37C1
Packed spring salad
94
CAZ, CTX, RFA, CIP
V70C1
Soybean sprouts
94
CAZ, CTX, ERY, RFA, TET, CIP
V71C3
Soybean sprouts
94
CAZ, CTX, ERY, RFA, CIP
V51C1
Lettuce
22
CAZ, CTX, RFA, TET, CIP, NA, NET, TMP/ STX
V26C1
Brocoli
26
CAZ, CTX, TET, CIP
V47C2
Strawberries
43
CAZ, CTX, TET, CIP, NA,
V16C1
Artichoke
46
CAZ, CTX, CIP
V17C1
Alfalfa sprouts
46
CAZ, CTX
V3C2
Green olives
55
CAZ, CTX, ERY, TET
V57C2
Celery
55
CAZ, CTX, ERY, TET
AMP ampicillin, CAZ ceftazidine, CTX cefotaxime, ERY erythromycin, RFA rifampin, TET tetracycline, CIP ciprofloxacin, NA nalidixic acid, NET netilmicin, TMP/STX trimethoprim/sulfamethoxazol, FUR nitrofurantoin
Antonie van Leeuwenhoek (2014) 105:413–421
food samples purchased in 15 different supermarkets located in the South of Spain. Each sample of the same food type was from a different supermarket. Isolates were cultivated routinely on brain heart infusion (BHI) broth (Scharlab, Barcelona, Spain) at 37 °C. Isolates were stored at -80 °C in BHI containing 20 % glycerol. MLST analysis The isolates were analyzed by MLST for E. faecium (http://efaecium.mlst.net; Homan et al. 2002) by using primer sequences available at http://efaecium.mlst.net/ misc/info.asp. The allelic profiles of isolates were obtained by sequencing internal fragments of seven housekeeping genes: adk (adenylate kinase), atpA (ATP synthase, alpha subunit), ddl (D-alanine: D-alanine ligase), gdh (glucose-6-phosphate dehydrogenase), gyd (glyceraldehyde-3-phosphate dehydrogenase), purK (phosphoribosylaminoimidazol carboxylase ATPase subunit), and pstS (phosphate ATP-binding cassette transporter). PCR conditions for all amplification reactions were as follows: initial denaturation at 94 °C for 3 min; 35 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; and extension at 72 °C for 5 min. Reactions were performed in 50 ll volumes with buffers and Taq polymerase. PCR products were purified with ExoSAP-IT (USB Europe GmbH) and sequenced with the respective primers in a CEQ 2000 XL DNA Analysis System (Beckman Coulter, CA). For every isolate, each gene was amplified at least two times and sequenced with the specific forward or reverse primer at least three times. The obtained sequences were submitted to the E. faecium MLST database (http://www. mlst.net) for assignment of alleles at each locus and a sequence type. Cluster analysis of the data was performed using the MLST database and the e-BURST algorithm. Sequence types of isolates are defined by the allelic profile at these seven loci, with each unique combination of alleles assigned a distinct sequence type number and the sequence types (STs) of all isolates were determined. Determination of antibiotic resistance Antibiotic resistance determination was performed by the disk diffusion method as described by the Clinical and Laboratory Standards Institute (CLSI 2011) on Mueller–Hinton agar (Scharlab, Barcelona, Spain),
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choosing antibiotics that are widely used or frequently included in studies of antimicrobial resistance in enterococci. Ampicillin (AMP, 30 lg), ceftazidine (CAZ, 30 lg), cefotaxime (CTX, 30 lg), imipenem (IPM, 10 lg), erythromycin (ERY, 15 lg), tetracycline (TET, 30 lg), ciprofloxacin (CIP, 5 lg), nalidixic acid (NA, 30 lg), netilmicin (NET, 30 lg), trimethoprim/ sulfamethoxazole (TMP/STX, 25/75 lg), nitrofurantoin (FUR, 300 lg) were from Biome´rieux (Madrid, Spain). Vancomycin (VAN, 30 lg), chloramphenicol (CMP, 30 lg) and rifampin (RIF, 5 lg) were from BBL (Madrid, Spain). The levels of antibiotic resistance for ampicillin (GlaxoSmithKline, Madrid), ceftazidime and cefotaxime (borth from Normon, Madrid) were also determined by broth microdilution tests on Mueller– Hinton broth following CLSI guidelines and using antibiotic concentrations of 2, 4, 6, 8, 12, 16, 32 and 64 mg/l. Assays were performed in 96-well microtiter plates. Optical density at 595 nm was recorded at time 0 and 24 h of incubation at 37 °C with an iMark Microplate Reader (BioRad, Madrid). Determination of biocide tolerance Benzalkonium chloride (BC), cetrimide (CT), hexadecylpyridinium chloride (HDP), didecyldimethylammonium bromide (AB), chlorhexidine (CH), triclosan (TC) and hexachlorophene were from Sigma-Aldrich (Madrid, Spain). Benzalkonium chloride commercial solution contained 50 % (w/v) of the active compound. Triclosan and hexachlorophene were dissolved (10 % w/v) in 96 % ethanol. All other biocides were dissolved aseptically in sterile distilled water at final concentrations of 5 % (CH and HDP) or 10 % (CT), and stored at 4 °C for B7 days. Poly-(hexamethylene guanidinium) hydrochloride (PHMG) solution (containing 7.8 % of PHMG, by weight) was a kind gift of Oy Soft Protector Ltd (Espoo, Finland). The commercial sanitizers P3 oxonia (25–35 % hydrogen peroxide, 0.83–2.5 N acetic acid and 0.26–0.66 N peracetic acid) and P3 topax 66 (2–5 % sodium hydroxide, 2–5 % sodium hypochlorite and 2–5 % alkylamine) were obtained from ECOLAB (Barcelona, Spain). Minimal inhibitory concentrations (MICs) to biocides were determined by the microbroth dilution method on 96-well bottom microtiter plates (Becton–Dickinson Labware, Franklin Lakes, NJ). Briefly, serial dilutions of biocides were incubated with bacterial suspensions adjusted to 5 9 105 CFU/ml in Mueller–Hinton Broth
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(Scharlab). Growth controls (inoculated growth medium without antimicrobials) and sterility controls (non-inoculated medium supplemented with antimicrobials) were included. Microtiter plates were incubated at 37 °C and readings were performed after 24 h of incubation by optical density (OD 595 nm) determination in an iMark Microplate Reader. MICs were determined as the minimum biocide concentration that achieved complete growth inhibition.
Results and discussion The 22 E. faecium isolates from fresh produce investigated comprised 7 different MLST types (STs 22, 26, 43, 46, 55, 94 and 296; Table 1; Fig. 1). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). Other STs (46 and 55) were represented by two isolates (9.1 %) or just one isolate (STs 22, 26,
ST43
ST46 ST 55
ST 22
78
CC17
Fig. 1 Population snapshot of E. faecium. The entire E. faecium MLST database is displayed as a single eBURST diagram. STs (nodes in the diagram) are labeled and STs differing in only one allele (i.e. single locus variants) are connected by arcs. Colors indicate predicted primary (blue) and
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43). The STs most represented in E. faecium from fresh produce only have a few representatives in the MLST database, most of them from clinical sources. For example, ST 296 includes two strains from hospitalized patients, one from a cat belly and one probiotic strain. The closest STs with six alleles in common (345, 694 and 695) include strains from blood of a hospitalized patient and from non-hospitalized persons. ST 94 is closely related to 12 additional STs and includes strains that were isolated from faeces of healthy people and from hospitalized patients. ST 22 is highly represented in the MLST database and includes strains from hospitalized patients (four of them being vancomycin-resistant), two vancomycin-resistant strains from cheese and two strains from pig faeces. ST 22 is strongly related (6 common alleles) to 24 other STs. ST 26 includes strains from chicken and turkey (VanA) faeces, hospitalized patients and a non-specified source.
ST 26
ST 94 ST 296
secondary (yellow) founders. The areas of each of the circles indicate the prevalence of the ST in the input data. The STs found in this study and the STs 17, 18 and 78 belonging to clonal complex 17 (CC17) are marked by arrows
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ST43 includes strains from calf faeces and from meat. Its two closest STs (172, 187) include VanA isolates from calf faeces. ST 46 includes one strain from a hospitalized patient. Its closest STs (69, 161 and 704) include isolates from river water and faeces from nonhospitalized persons. There are few studies on MLST typing of enterococci from vegetable foods. In one study on E. faecalis, Grande Burgos et al. (2009) reported that most isolates belonged to ST 168. However, one isolate belonged to ST 9 of the previously-described clonal complex (CC) 9 which is frequently associated with hospital environments. In a recent study, E. faecium isolates from the rhizosphere were assigned to STs 21, 606, 613, 614 and 615, demonstrating new genetic lineages among rhizosphere isolates (Klibi et al. 2012). Since many of the isolates included in the MLST database originate from clinical sources, it is important to also investigate isolates from other sources such as the environment or fresh produce in order to achieve a more complete picture of the population diversity and relatedness of E. faecium isolates. The antibiotic resistance of isolates is shown in Table 1. None of isolates was resistant to vancomycin. All isolates were sensitive to imipenem and only one isolate (V39C4 from ST 296) was resistant to ampicillin (MIC 32 mg/l). E. faecium has a high capacity to acquire high levels of ampicillin resistance under selective pressure of this antibiotic (Rice et al. 2001). Ampicillin resistance is now recognized as a marker for strains belonging to CC17 (Top et al. 2008). All isolates were resistant to cefotaxime and ceftazidine. MICs for cefotaxime were[64 mg/l for most isolates, except V11C1 (32 mg/l) or V36C1 (64 mg/l). MICs for ceftazidine ranged from 16 mg/l (isolate V11C1) to 32 mg/l (V3C2, V50C1), 64 mg/l (V38C1) or [64 mg/l (rest of isolates). In enterococci, betalactam resistance is mediated by Pbp5 in cooperation with class A PBPs (Rice et al. 2001). However, resistance to ampicillin and cephalosporins can be dissociated by the deletion of the class A pbp genes, pbpF and pbpA, indicating a complex mechanism of beta-lactam resistance in this bacterium (Rice et al. 2009). Enterococci are intrinsically resistant to cephalosporins (Murray 1990) and cephalosporin administration may select for enterococcal infections (Linden and Miller 1999). Netilmicin resistance was detected in four isolates in the present study but
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according to previous data (Abriouel et al. 2008), all isolates were sensitive to gentamicin. Aminoglycosides such as netilmicin or gentamicin are recommended for the treatment of E. faecalis infections, especially in severe and bacteremic infection (Dube´ et al. 2012). In a previous study, we reported that erythromycin and rifampicin were the most frequent antibiotic resistance traits in E. faecium from vegetable foods, followed by ciprofloxacin and tetracycline (Abriouel et al. 2008). These results were also confirmed in the present study. The use of antibiotics such as erythromycin or tetracycline in animal husbandry and agriculture, and the spread of resistant strains through polluted irrigation waters, animal faeces or insects seem to be contributing factors for the presence of antibioticresistant enterococci in the environment (Hollenbeck and Rice 2012; Sa´nchez Valenzuela et al. 2012; Furtula et al. 2013; Micallef et al. 2013). Enterococci carrying antibiotic resistance traits have been frequently found in tomatoes, as well as in the irrigation waters and soil (Micallef et al. 2013). Ground distance and type of farm (small vs. large farms) influenced the incidence of enterococci in tomatoes. It has been suggested that, unlike large-scale farms that fertilized exclusively with chemical nutrients, small-scale farms occasionally used poultry litter for fertilization. The current recommended composting times for poultry litter may not be long enough to eliminate all enterococci (Graham et al. 2009). Enterococci from our study were isolated from a variety of vegetable foods bought at 15 different supermarkets and obtained by different cultivation practices. Most of them grow a short distance from the ground (such as lettuce, broccoli, endives, celery or asparagus), deep in the ground (such as beet or potato) or in hydroponic cultures (such as sprouts). Therefore, contamination of many of these foods by enterococci from environmental sources seems very likely. Many of these foods are sold without being washed (like for example whole lettuce or endives), while others may be washed and processed, such as the ready to eat salads. Conventional chlorine washing steps may be insufficient to inactivate enterococci in salads, as shown in a recent study (Campos et al. 2013). Chlorine disinfection is widely used on fresh produce. In addition, many other biocidal substances are used by the food industry for cleaning and disinfection practices (McDonnel and Russell 1999; Cerf et al.
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active biocide among quaternary ammonium compounds, requiring 500 mg/l or even 750 mg/l for two isolates. Previous studies have reported MICs for BC of 2–4 mg/l in E. faecium isolates from dust in breeding pigs facilities (Braga et al. 2013), or between 2 and 16 mg/l in E. faecium isolates from livestock (Aarestrup and Hasman 2004). Sidhu et al. (2002) proposed that Enterococcus sp. isolates with MICs for BC \30 mg/l should be considered sensitive to this biocide, while 30–50 mg/l was considered as low-level resistance and [50 mg/l was considered as high-level resistance. In another study, between 92.5 and 100 % of enterococci from foods, animals or clinical sources were inhibited by 25 mg/l BC, and most isolates also were inhibited by 25 mg/l CT or 2.5 mg/l HDP (Valenzuela et al. 2013). All isolates from the present study were inhibited at 75 mg/l of the biguanide CH, while MICs for the
2010). Food processing facilities and utensils such as transport containers, boxes, conveyor belts, cutting and peeling machines, floor drains or storage rooms where organic matter accumulates frequently become sources of bacterial contamination and need to be disinfected periodically. These may be potential sources of enterococci exposed to different types of biocides that may then enter the food chain. The results obtained in the present study on biocide tolerance of E. faecium isolates from fresh produce are shown in Table 2. Among the quaternary ammonium compounds, the most active biocide was HDP, which inhibited most isolates at 1 mg/l. For BC, 40.9 % of isolates were inhibited at 12.5 mg/l, while others required 25 or 37.5 mg/l, and one isolate was inhibited only at 50 mg/l (Table 2). Most isolates required 75 mg/l CT for inhibition but some were also inhibited at 25 mg/l. AB was the least
Table 2 Biocide tolerance in E. faecium isolates from fresh produce Biocide concentration (mg/l)
Biocide MIC distribution (%) BC
CT
HDP
1
95.46
2
4.54
AB
CH
PHMG
TC
5
CF
OX
TOP
95.45
100
22.73
8
27.27
10 12.5
13.63 40.91
20
40.91
25
22.73
37.5
31.81
50
4.55
31.82 4.54
63.64
60 75
31.82 63.64
100
100 500
90.91
27.28
750 0.25a
9.09
72.72
0.5a 0.75a
4.55
Minimal inhibitory concentrations and their corresponding percentages of isolates are shown Ranges of biocide concentrations recommended by manufacturers: BC, 0.05–0.2 %; CT, 0.1–0.5 %; HDP, up to 0.8 %; AB, 0.01–0.1 %; CH, 0.01–4.0 %; PHMG, 0.1–0.2 %; TC, 0.3–2 %; CF, up to 0.1 %; OX and TOP, 0.05 to 5 % BC benzalkonium chloride, CT cetrimide, HDP hexadecylpyridinium chloride, AB didecyldimethylammonium bromide, CH chlorhexidine, PHMG poly-(hexamethylen guanidinium) hydrochloride, TC triclosan, CF hexachlorophene OX P3 oxonia commercial solution, TOP P3 topax 66 commercial solution a
Biocide concentration is expressed as % of commercial solution (v/v)
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polyguanide PHMG ranged from 8 to 60 mg/l (Table 2). Reported MICs for CH from other studies on enterococci were of 2 mg/l or lower (Beier et al. 2008), 2–4 mg/l (Braga et al. 2013), 4–6 mg/l (Suller and Russell 1999) or even up to 2,500 mg/l in clinical isolates (Valenzuela et al. 2013). Formulations containing biocides such as BC, CH and PHMG are used for sanitizing the surfaces of utensils and instruments in the food industry at concentrations of 0.05–0.2, 0.01–0.02, and 0.1–0.2 % of the active ingredient, respectively (Ueda and Kuwabara 2007). CH is also frequently used in personal care and hand hygiene products at concentrations of 2–4 % (Kampf and Kramer 2004). HDP was approved in 2004 by the USA Food and Drug Administration for decontaminating raw poultry at a working concentration not exceeding 0.8 % by weight (Food and Drug Administration 2004). Among the bisphenols tested, TC required concentrations of 500–750 mg/l for inhibition, while CF was active at lower concentrations but it had a broader range of MICs, from 5 to 50 mg/l (Table 2). Other studies on enterococci have reported MICs for TC from 3 to 4 mg/l (Suller and Russell 1999), 8 mg/l (Beier et al. 2008) or even higher than 250 mg/l (Valenzuela et al. 2013). TC is now incorporated in a wide range of products, such as dishcloths, food boxes, toothbrushes, washing-up liquid and hand-washing gels, plastics, chopping boards, chopsticks, pizza cutters, food storage containers, garbage bags, or kitty litter, among others, at recommended concentrations that may range from 0.3 to 1 % (Yazdankhah et al. 2006) or even 1–2 % (Kampf and Kramer 2004). The commercial solutions P3 oxonia and P3 topax 66 were active on E. faecium isolates at final concentrations of 0.25–0.75 % by volume (Table 2), which is in the range of their recommended use concentrations of 0.05–5 % (Blakistone et al. 1999). There is also a concern that exposure to biocides and biocide tolerance may co-select for antibiotic resistance (Ortega-Morente et al. 2013). However, previous studies have shown that antibiotic-resistant enterococci are sensitive or moderately resistant to biocides such as BC, TC or CH (Suller and Russell 1999; Fraise 2002; Aarestrup and Hasman 2004; Beier et al. 2008). In the present study, those isolates showing greater numbers of antibiotic resistance traits had lowest MICs for biocides, as in the case of isolate V12C1 (which had lowest MICs for BC, CT, CF, AB, TC and HDP) and isolate V51C1 (with lowest MICs for BC, CT and HDP). These results
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may be an indication that antibiotic resistance and biocide tolerance are not related. Another concern about enterococci is the presence of virulence factors (Gilmore et al. 2013). According to our previous study, the incidence of virulence factors among E. faecium isolates from vegetable foods was much lower compared to clinical isolates, although 45.5 % of isolates carried the gelatinase gene gelE (Abriouel et al. 2008). None of isolates showed betahaemolytic activity, and none carried the complete set of cytolysin genes. While 54.5 % of isolates carried cylB, only 4.5 % carried cylA or cylM. Other virulence genes, such as aggregation substance (agg) and enterococcal surface protein (esp) were detected at low frequencies (18.2 and 27.3 %) compared to clinical isolates, while other genetic determinants frequently found in clinical isolates such as the collagen adhesin (ace) and the E. faecium endocarditis gene (efafm) were not detected (Abriouel et al. 2008). These previous results are in agreement with the observed lack of resistance to antibiotics such as vancomycin or ampicillin typically found in clinical isolates and also with the obtained MLST data, suggesting that the studied isolates belong to subpopulations not related to nosocomial strains. In conclusion, results from the present study indicate that the E. faecium isolates from the fresh produce analysed are not related to the clinically relevant clonal complex CC17. Some STs (especially ST 296 but also ST 94) seem to be more common in different types of fresh produce. Antimicrobial resistance seems to be widely disseminated among the studied enterococci from fresh produce, but their biocide tolerance levels fall below the recommended use concentrations of biocides. Acknowledgments This work was supported by research project P08-AGR-4295 (Junta de Andalucı´a CICE, FEDER) and University of Jae´n (Plan Propio). We also acknowledge the Campus de Excelencia Agroalimentario CeiA3. Conflict of interest The authors declare that they have no conflict of interest.
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