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Food Microbiology 42 (2014) 61e65

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Food Microbiology

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Antimicrobial resistance of Listeria monocytogenes and Listeria innocuafrom meat products and meat-processing environment

Diego Gómez, Ester Azón, Noelia Marco, Juan J. Carramiñana, Carmina Rota,Agustín Ariño*, Javier YangüelaDepartment of Animal Production and Food Science, Veterinary Faculty, University of Zaragoza, c/Miguel Servet 177, 50013 Zaragoza, Spain

a r t i c l e i n f o

Article history:Received 13 July 2013Received in revised form17 February 2014Accepted 18 February 2014Available online 28 February 2014

Keywords:Listeria monocytogenesListeria innocuaAntimicrobial resistanceReady-to-eat meat productsProcessing environment

* Corresponding author. Tel.: þ34 876554131; fax:E-mail address: aarino@unizar.es (A. Ariño).

http://dx.doi.org/10.1016/j.fm.2014.02.0170740-0020/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

A total of 336 Listeria isolates from ready-to-eat (RTE) meat products and meat-processing environments,consisting of 206 Listeria monocytogenes, and 130 Listeria innocua isolates, were characterized by discdiffusion assay and minimum inhibitory concentration (MIC) values for antimicrobial susceptibilityagainst twenty antimicrobials. Resistance to one or two antimicrobials was observed in 71 L. mono-cytogenes isolates (34.5%), and 56 L. innocua isolates (43.1%). Multidrug resistance was identified in 24Listeria isolates, 18 belonging to L. innocua (13.9%) and 6 to L. monocytogenes (2.9%). Oxacillin resistancewas the most common resistance phenotype and was identified in 100% Listeria isolates. A mediumprevalence of resistance to clindamycin (39.3% isolates) and low incidence of resistance to tetracycline(3.9% isolates) were also detected. Listeria isolates from RTE meat products displayed higher overallantimicrobial resistance (31.3%) than those from the environment (13.4%). All the strains assayed weresensitive to the preferred antibiotics used to treat listeriosis. Results showed that although antimicrobialresistance in L. monocytogenes still occurs at a low prevalence, L. innocua can form a reservoir of resis-tance genes which may transfer between bacterial species, including transference to organisms capableof causing disease in humans.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

In the 1980s, Listeria monocytogenes infection (listeriosis) wasrecognized as a foodborne illness which can lead to invasive diseaseduring vulnerable stages of life. Older adults, pregnant women andpersons with immunocompromising conditions are at higher riskfor Listeria bacteremia and meningitis, which can be fatal (Rocourtand Buchrieser, 2007; Drevets and Bronze, 2008). Outbreaks andsporadic cases of listeriosis have been associated with contamina-tion of various food items including milk, soft cheese, meat andmeat products, vegetables, seafood products, and ready-to-eatfoods (Codex Alimentarius, 2007).

The average annual incidence of foodborne infections caused byL. monocytogenes is low in comparison with other pathogens suchas Salmonella, Campylobacter and pathogenic Escherichia coli,including Verotoxigenic E. coli (VTEC). However, L. monocytogenes isconsidered as an important foodborne pathogen due to its highmortality, with a case-fatality rate of up to 30% (Lecuit and Leclercq,

þ34 976761612.

2012). During the year 2010, the number of human listeriosis casesreported in Europe was 1,601, 3.2% fewer than in 2009. The mostwidely affected population group was people over the age of 65years (60.2% of cases) and themortality ratewas 17.0% (EFSA, 2012).

L. monocytogenes, a bacterium which is ubiquitous in the envi-ronment, is naturally susceptible to a range of antibiotics that act onGram-positive bacteria (Charpentier and Courvalin, 1999). TheComité de l’Antibiogramme de la Société Française de Microbiologie(CA-SFM) (CA-SFM, 2010) and the National Reference Center forListeria (NRCL) (Lecuit and Leclercq, 2012) indicated that humanstrains of L. monocytogenes are sensitive to a wide range of antibi-otics that include penicillin, ampicillin, amoxicillin, gentamicin,erythromycin, tetracycline, rifampicin, co-trimoxazole, vancomycinand imipenem. However, most strains of L. monocytogenes shownatural resistance to current fluoroquinolones and cephalosporins,especially third and fourth generation, such as cefotaxime andcefepime, and also to fosfomycin, oxacillin and licosamides. Clini-cians typically use aminopenicillins (e.g., ampicillin or amoxicillin)in combinationwith an aminoglycoside, such as gentamicin, for thetreatment of invasive infections. In cases where reduced sensitivityor resistance to beta-lactams is encountered, a number of agentsactive against Gram-positive bacteria may be used, though

Table 1Strains of Listeria spp. used in the drug resistance study.

Source Origin Sample type L. monocytogenes L. innocua

Food industry Surface Stainless steel 53 29HMWPa 9 1Conveyor belt 10 1Subtotal 71 31

RTE meatproduct

Cooked 21 12Raw-cured 53 42Dry-curedand ripened

9 8

Marinated 2 0Subtotal 85 62

Marketsamples

RTE meatproduct

Cooked 35 25Raw-cured 10 10Dry-curedand ripened

4 2

Subtotal 49 37Total 206 130

a High molecular weight polyethylene.

D. Gómez et al. / Food Microbiology 42 (2014) 61e6562

cotrimoxazole is generally regarded as the second-choice thera-peutic option (Lecuit and Leclercq, 2009).

On the other hand, the application of antimicrobial agents isnow seriously jeopardized by the emergence and spread of mi-crobes that are resistant to affordable and effective first choiceantibiotics. Increasing global trade and travel favor the spread ofantimicrobial resistance between countries and continents.Therefore, antimicrobial resistance is a global public health concern(Doyle et al., 2013). One action to address threats posed by anti-microbial resistance includes the monitoring programs forantimicrobial-resistant microbes that integrate clinical settings andalong the food chain. This is useful to identify trends in develop-ment and persistence of antimicrobial resistance among selectfoodborne pathogens. Studies performed by the NRCL with humanstrains and, to a lesser extent, with foodstuff strains do not revealan increase in resistance to antibiotics by the circulating strains ofL. monocytogenes (Morvan et al., 2010; Granier et al., 2011).

However, since the isolation of the first multi-resistant strain ofL. monocytogenes in 1988, interspecies variation in antimicrobialsusceptibilities has been reported in Listeria species (Charpentieret al., 1995; Margolles et al., 2001). The pattern of susceptibilitybetween L. monocytogenes and Listeria innocua is important owingto the fact that both species are very often found in the same food orprocessing environment (Gómez et al., 2012). Balsalobre andHernández-Godoy (2004) have suggested that species of Listeria,such as L. innocua, and other Gram-positive bacteria which are veryoften present in meat and meat products, show frequent resistanceto antibiotics, with the possibility of a transfer of genetic informa-tion from one species to another through various mechanisms. Thistransfer has been demonstrated in vitro for streptomycin, erythro-mycin and chloramphenicol. Additionally, considering the highmortality rate of listeriosis, it is important to ensure the effective-ness of antimicrobials for listeriosis and monitor the emergence ofantimicrobial-resistant Listeria strains.

The purpose of this study was to determine the resistancepattern of Listeria strains isolated from RTE meat products andfood-contact surfaces in meat industries to various antibiotics thatare widely used in human and veterinary medicine.

2. Material and methods

2.1. Bacterial isolates

A total of 336 Listeria isolates from ready-to-eat (RTE) meatproducts and food-processing environments, including 206 L.monocytogenes, and 130 L. innocua isolates, were characterized byantimicrobial susceptibility tests. The strains were isolated be-tween May 2009 and April 2012 according to the ISO 11290-1 (ISO11290-1: 1996/Amd 1, 2004a) and ISO-11290-2 (ISO 11290-2: 1998/Amd 1, 2004b) standards as described in detail elsewhere (Gómezet al., 2012). They were pre-identified (b-hemolysis and fermen-tation of rhamnose, xylose and mannitol) and finally identifiedwith the gallery API-Listeria.

Table 1 shows the origin of the Listeria isolates used in this study.Of the 336 strains investigated, 103 (72 L. monocytogenes and 31L. innocua) were isolated from environmental samples taken from32 Spanish meat industries located across NE and SW Spain. Thestrains were isolated from food-contact surfaces made of stainlesssteel, high molecular weight polyethylene (HMWP) and polyvinylchloride (PVC) conveyor belts. Another set of 147 strains (85L. monocytogenes and 62 L. innocua) were isolated from the RTEmeat products produced within the sampled industries. Theremaining 86 strains of Listeria (49 L. monocytogenes and 37L. innocua) were isolated from commercially available RTE meatproducts purchased in butcher’s shops, supermarkets and

hypermarkets in NE Spain (Table 1). The Listeria strains were kept incryovials (Vibakstore, Nirco S.L., Barcelona, Spain) and storedat �80 �C.

2.2. Antimicrobial susceptibility testing: disk diffusion method andMIC values

Antimicrobial susceptibility testing of Listeriawas performed bythe disc diffusion method as recommended by the EuropeanCommittee on Antimicrobial Susceptibility Testing (EUCAST, 2012).Mueller-Hinton agar plates were used (Oxoid, Hampshire, UK) withincubation at 35 �C for 24 h. The inoculum was standardized byspectrophotometry (Spectronic 20, Bausch&Lomb, Rochester, N.Y.,USA) inoculating the plates with a concentration of approximately108 cfu/ml using a cotton swab.

The following 20 antimicrobials (and disk load), including thoseused to treat human listeriosis, were tested: amoxicillin-clavulanicacid (30 mg; AMC), ampicillin (10 mg; AMP), ciprofloxacin (5 mg; CIP),clindamycin (2 mg; DA), clarithromycin (15 mg; CLR), chloram-phenicol (30 mg; C), gentamicin (10 mg; CN), imipenem (10 mg; IPM),levofloxacin (5 mg; LEV), linezolid (30 mg; LZD), meropenem (10 mg;MEM), moxifloxacin (5 mg; MXF), oxacillin (1 mg; OX), penicillin G(10 mg; P), rifampicin (30 mg; RD), trimethoprimesulfamethoxazole[1.5 mg-23.5 mg; SXT], teicoplanin (30 mg; TEC), tetracycline (30 mg;TE), tigecycline (15 mg; TGC) and vancomycin (30 mg; VA). Addi-tionally, in the case of L. innocua, resistance to five more antibioticswas assayed: erythromycin (15 mg; E), minocycline (30 mg; MH),streptomycin (10 mg; S), sulfamethoxazole (25 mg; RL) andtrimethoprim (5 mg; W). The following quality control strains wereincluded with each batch: E. coli ATCC 25922, Staphylococcus aureusATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Enterococcusfaecalis ATCC 29212.

The diameters of growth inhibition zones were measured andinterpreted according to the breakpoints recommended by theEUCAST (2013) for the various types of antibiotics, and the strainswere classified as sensitive, intermediate (reduced susceptibility)or resistant.

There were 62 L. monocytogenes and 85 L. innocua isolates forwhich the interpretation of the disc diffusion method was unclearfor six antibiotics. These strains were confirmed by determining theminimum inhibitory concentrations (MIC) by graded-concentrationantibiotic strips (M.I.C.E. strips; Oxoid). Simply, 0.5 McFarlandinoculum of the Listeria isolates was swab-spread over MuellereHinton agar plates and, then, M.I.C.E. strips were aseptically placedon the dried surface within 15 min. Plates were incubated at 35 �C

Table 2Antibiotics and strains of Listeria spp. studied using the M.I.C.E. strips.

Concentration(mg/ml)

Abbreviation L. monocytogenes L. innocua

Ciprofloxacin 32e0.002 CIP 23 44Clindamycin 256e0.015 DA 19 21Linezolid 256e0.015 LZD 1 1Meropenem 32e0.002 MEM 3 e

Penicillin G 32e0.002 P 15 7Tetracycline 256e0.015 TE 1 12Total 62 85

Table 4Antibiotic-resistant Listeria monocytogenes isolated from meat products and food-processing environments.

Antibiotic Isolated from Reference

Ampicillin Retail meats Walsh et al. (2001)Meat products Yücel et al. (2005)

Chloramphenicol Pork sausage Rota et al. (1996)Meat products Yücel et al. (2005)Poultry meat Miranda et al. (2008)

Clindamycin Poultry carcasses Antunes et al. (2002)Ciprofloxacin Foodstuffs and processing

environmentConter et al. (2009)

Poultry meat Alonso-Hernandoet al. (2012)

Erythromycin Pork sausage Rota et al. (1996)Poultry meat Miranda et al. (2008)

Gentamicin Poultry meat Alonso-Hernandoet al. (2012)

Linezolid Foodstuffs and processingenvironment

Conter et al. (2009)

Rifampicin Foodstuffs and processingenvironment

Conter et al. (2009)

Poultry meat Alonso-Hernandoet al. (2012)

Streptomycin Poultry carcasses Antunes et al. (2002)Tetracycline Pork sausage Rota et al. (1996)

Foodstuffs and processingenvironment

Conter et al. (2009)

Raw meat and retail foods Pesavento et al. (2010)Raw chicken and RTE chickenproducts

Osaili et al. (2011)

Ducks Adzitey et al., 2013Trimethoprim/

sulfamethoxazoleMeat products Yücel et al. (2005)Poultry meat Miranda et al. (2008)

Vancomycin Foodstuffs and processingenvironment

Conter et al. (2009)

Raw meat and retail foods Pesavento et al. (2010)

D. Gómez et al. / Food Microbiology 42 (2014) 61e65 63

for 24 h and the interpretation of results was made according to themanufacturer’s protocol (Table 2).

3. Results

This study has determined the resistance to 20 antibiotics of 206strains of L. monocytogenes and the resistance to 25 antibiotics of130 strains of L. innocua, both species isolated from RTE meatproducts and food-contact surfaces from meat processing plants.Although most isolates were antimicrobial susceptible, resistancephenotypes were detected against six antimicrobials (see Table 3).

Species variation in antimicrobial susceptibilities was observed,as resistance to one or two antimicrobials was detected in 71 L.monocytogenes isolates (34.5%) as compared to 56 L. innocua iso-lates (43.1%). The rankings of multi-resistance showed that 13.9% ofL. innocua isolates showed resistance to three or more antibiotics,while only 2.9% isolates of L. monocytogenes were multi-resistant.Listeria isolates from RTE meat products displayed higher overallantimicrobial resistance (31.3%) than those from the environment(13.4%).

Resistance to the b-lactam oxacillin was the most commonresistance phenotype and was identified in 100% of Listeria strainsof both species. It is worth noting the prominent appearance ofdrug resistance to clindamycin, which reached prevalence levelsfrom 35 to 46.2% in L. monocytogenes and L. innocua, respectively.Also, one strain of L. monocytogenes (0.5%) and twelve strains ofL. innocua (9.2%) were found to be resistant to tetracycline. Withregard to fluoroquinolones, intermediate susceptibility to cipro-floxacin was found for one strain of the pathogenic species (0.5%)and twelve strains of L. innocua (9.2%). One strain of L. innocua(0.8%) showed intermediate susceptibility to chloramphenicol. All

Table 3Percentages of sensitivity (S), intermediate susceptibility (I) and resistance (R) to theantimicrobials tested on the 336 strains of Listeria spp. isolated from RTE meatproducts and food-contact surfaces.

Antibiotics L. monocytogenes (n ¼ 206) L. innocua (n ¼ 130)

Sa I R Sa I R

b-LactamsOxacillin 100 100Penicillin G 96.1 3.9 94.6 5.4

TetracyclinesTetracycline 99.5 0.5 90.8 9.2

FluoroquinolonesCiprofloxacin 99.5 0.5 90.8 9.2

AmphenicolsChloramphenicol 100 99.2 0.8

LincosamidesClindamycin 65.0 35.0 53.8 46.2

a All the strains of Listeria spp. proved sensitive to the other antibiotics:amoxicillin-clavulanic acid, ampicillin, clarithromycin, gentamicin, imipenem, lev-ofloxacin, linezolid, meropenem, moxifloxacin, rifampicin, trimethoprimesulfa-methoxazole, teicoplanin, tigecycline and vancomycin.

Listeria strains were highly sensitive to the preferred antibioticsused to treat listeriosis, such as ampicillin and amoxicillin, with orwithout gentamicin, or trimethoprimesulfamethoxazole (co-tri-moxazole) as a second choice. However, special mention must bemade of the reduced susceptibility of eight strains ofL. monocytogenes (3.9%) and seven strains of L. innocua (5.4%) topenicillin G.

Regarding multi-resistance, six strains of L. monocytogenes(2.9%) showed resistance to two antibiotics (clindamycin andoxacillin) and intermediate susceptibility to penicillin G. However,ten L. innocua strains (7.7%) revealed resistance to three antibiotics(clindamycin, oxacillin, and tetracycline), and four of them showedintermediate susceptibility to penicillin G. Another eight strainsproved resistant to clindamycin and oxacillin and five of said strainsshowed intermediate susceptibility to ciprofloxacin and the rest topenicillin G.

4. Discussion

Ever since the first strain of L. monocytogenes with antibioticresistance was isolated in 1988, resistance to one or more antimi-crobials has been found in strains isolated in foodstuffs and in theenvironment, as well as of human origin. A list of L. monocytogenesantibiotic-resistant strains isolated from meat products and food-processing environment is presented in Table 4. Given theincreasing number of antibiotic-resistant L. monocytogenes strainsbeing isolated around the world, it appears that this pathogen israpidly acquiring awide variety of antibiotic resistance genes, manyof which may come from the commensal organisms found in foodsand food-growing and food-processing areas (Lungu et al., 2011).

D. Gómez et al. / Food Microbiology 42 (2014) 61e6564

This finding is a cause for concern and suggests that resistance inclinical human isolates may emerge in the near future.

Antibiotic resistance in L. monocytogenes is chiefly caused bymobile genetic elements such as self-transferable plasmids, mobi-lizable plasmids, and conjugative transposons. The plasmid pIP501,which has a broad host range and confers resistance to chloram-phenicol, macrolides, lincosamides, and streptogramins, was thefirst reported to be transferable by conjugation to L. monocytogenes,replicate within Listeria as well as be transferable between speciesof Listeria (Pérez-Diaz et al., 1982). Lemaitre et al. (1998) discoveredthat 9% of L. monocytogenes and 3% of L. innocua strains tested wereresistant to multiple antimicrobials and that these resistance phe-notypes were transferable among Listeria spp., suggesting they areencoded in mobile plasmids. In a recent study, a multidrug resis-tance plasmid (called pDB2011) isolated from a foodborneL. innocua strain, harbored the dfrD trimethoprim resistance genethat was 100% identical to dfrD found in a number ofL. monocytogenes isolates, showing a large potential for dissemi-nation as indicated by its host range of both Gram-positive andGram-negative bacteria (Bertsch et al., 2013).

It has been suggested that L. innocua strains constitute a reser-voir of antibiotic resistance transferable to L. monocytogenes(Bertrand et al., 2005). Li et al. (2007) detected the gene tet(M) thatconfers resistance to tetracyclines in antimicrobial-resistantL. innocua from bison that could potentially transfer resistance toL. monocytogenes, as well as Chen et al. (2010), who also found thegene tet(M) in Listeria from catfish. L. innocua and L. monocytogenesare very similar genetically so that the study of the closely related,but nonpathogenic L. innocua, accounts for a better understandingof the antibiotic resistance of L. monocytogenes (Milillo et al., 2012).

In the present study, the L. monocytogenes strain that wasresistant to tetracycline was isolated from a stainless steel surface,while the 12 strains of L. innocua that proved resistant to thisantibiotic came from cooked meat products. The incidence ofresistance to tetracycline is increasingly common among strains ofListeria spp. isolated from foodstuffs and the environment (seeTable 4). The effectiveness of tetracycline diminished in the pre-ceding decades owing to the widespread existence of resistancegenes, probably as a result of the prolonged and extensive use ofthese antimicrobials in human beings and as growth promoters inanimals.

An increase in the frequency of antibiotic resistance inL. monocytogenes since the 1990s has been observed for all majorclasses of antibiotics. In Spain, a study was conducted to comparethe evolution of antibiotic resistance profiles of L. monocytogenesisolates from poultry between 1993 and 2006 (Alonso-Hernandoet al., 2012). 37.2% of strains in 1993 and 96.0% in 2006 showedresistance to at least one antibiotic. Multi-resistance was lesscommon in 1993 than in 2006 (18.6% and 84.0%, respectively), andthe average number of antibiotics to which the strains wereresistant was lower in 1993 (1.6%) than in 2006 (4.2%). A signifi-cant increase in the percentage of resistant strains was observedbetween 1993 and 2006 for six different drugs: gentamicin,streptomycin, neomycin, enrofloxacin, ciprofloxacin andfurazolidone.

In the present study, Listeria isolates from RTE meat productsdisplayed higher overall antimicrobial resistance (31.3%) than thosefrom the environment (13.4%). Similarly, Kovacevic et al. (2013)indicated that antibiotic resistance was more commonly observedfor L. monocytogenes in RTE foods than in processing environmentsamples, describing a co-selection phenomenon in which repeatedexposure to sub-lethal concentrations of some antimicrobials (i.e.ciprofloxacin) may produce derivative strains possessing increasedtolerance to the respective selective agent as well as increasedtolerance to other antibiotics.

Clinicians typically use ampicillin, amoxicillin with or withoutgentamicin or trimethoprimesulfamethoxazole (co-trimoxazole)for the treatment of listeriosis. Resistance to these antibiotics is stillnot a problem for the first-choice therapeutic treatment for liste-riosis (EFSA, 2008). All the strains assayed in the present studywerehighly sensitive to the preferred antibiotics used to treat listeriosisand these results concurwith those found by Pesavento et al. (2010)in raw meat isolates, Wang et al. (2013) in retail raw foods, andLecuit and Leclercq (2012) in human strains.

Although L. monocytogenes strains with resistance to one ormore antibiotics have been isolated, overall resistance to antibioticscommonly used to treat listeriosis has rarely been observed.However, there is a rising threat from antimicrobial resistance sincethe bacteria can develop resistance mechanisms or acquire resis-tance by the transmission of genetic material from other bacterialspecies. Therefore, the presence of such resistance in other Listeriaspecies raises the possibility of future acquisition of resistance byL. monocytogenes.

Acknowledgments

This work was supported by the following projects: CarnisenusaCSD 2007-00016 (Consolider Ingenio 2010, MICINN, Spain), A01/2013 (Grupo Consolidado de Investigación, DGA) and ESF (Euro-pean Social Fund).

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