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Food Microbiology 30 (2012) 372e378

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Food Microbiology

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Occurrence and characterization of Listeria spp. in ready-to-eat retail foodsfrom Vancouver, British Columbia

Jovana Kova�cevi�c, Lili R. Mesak, Kevin J. Allen*

Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, 2205 East Mall, Vancouver, V6T 1Z4, British Columbia, Canada

a r t i c l e i n f o

Article history:Received 31 October 2011Accepted 15 December 2011Available online 2 January 2012

Keywords:Listeria monocytogenesReady-to-eat foodRetail foodVirulence riskInternalinAntimicrobial resistance

* Corresponding author. 218-2205 East Mall, VCanada V6T 1Z4. Tel.: þ1 604 822 4427; fax: þ1 604

E-mail address: allen12@mail.ubc.ca (K.J. Allen).

0740-0020/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.fm.2011.12.015

a b s t r a c t

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products inVancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recoveredL. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventionalmethods were used to recover Listeria spp. from deli meat (n ¼ 40) and fish (n ¼ 40) samples collectedfrom 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua,5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolatesencoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeriaspp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin,two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin wasseen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility toamikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeriisolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA,LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Listeria monocytogenes is an organism commonly associatedwith food-processing environments and ready-to-eat (RTE) foods.Although an infrequent cause of foodborne disease, it is linked todisproportionately high levels of morbidity and mortality (Clarket al., 2010; Weatherill, 2009). Its presence in RTE products isparticularly troublesome for vulnerable populations. This group ofpeople, including pregnant women and their foetuses, the reallyyoung and the elderly, is particularly susceptible to invasive liste-riosis, with mortality rates ranging between 20 and 40% (Clarket al., 2010; Public Health Agency of Canada, 2010). Evidencesuggests the risk to vulnerable populations may be even higher ifvirulent strains of L. monocytogenes in RTE foods are encountered(Chen et al., 2006; Gilmour et al., 2010).

In Canada, eight listeriosis outbreaks have been reported overthe years and have been linked to a variety of RTE foods (Clark et al.,2010; Health Canada, 2011). The most notable, however, wasthe 2008 nationwide outbreak associated with contaminated deli

ancouver, British Columbia,822 5143.

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meats that originated from a single food-processing facility (PublicHealth Agency of Canada, 2010), and resulted in 57 invasive liste-riosis cases and 23 deaths (Weatherill, 2009). The originatingsource of contamination was suspected to be a large commercialslicer harbouring L. monocytogenes (Weatherill, 2009). The facility’senvironmental sampling records showed the intermittent presenceof L. monocytogenes on two processing lines for almost a year priorto the outbreak. Similar scenarios have been reported in otherlisteriosis outbreaks where L. monocytogenes in the processingenvironment led to contamination of RTE products (CDC 2002;Mead et al., 2006; Olsen et al., 2005). It is well established thatfood product contamination is associated with food-processingenvironments harbouring L. monocytogenes and subsequent post-processing transfer to finished products (Lappi et al., 2004;Lundén et al., 2002; Olsen et al., 2005; Tompkin, 2002). Numerousstudies have focused on the prevalence of Listeria spp. in productionenvironments and contamination patterns in these facilities (Barroset al., 2007; Chasseignaux et al., 2002, 2001; Eklund et al., 1995;Norton et al., 2001). Strains of L. monocytogenes capable of per-sisting in food-processing environments for up to 12 years andintermittently contaminating products have been reported (Holahet al., 2004; Lundén et al., 2002; Olsen et al., 2005; Senczek et al.,2000). Retail establishments of RTE foods, however, have received

J. Kova�cevi�c et al. / Food Microbiology 30 (2012) 372e378 373

less attention and consequently fewer data examining prevalenceare available.

Canadian data on the presence of L. monocytogenes in retailRTE foods have varied across studies (Bohaychuk et al., 2006; Dillonet al., 1994; Farber, 1991, 2000). As a result, contradicting messageshave been conveyed regarding the safety of Canadian RTE foods. In1991, Farber (Farber, 1991) reported results of a limited samplingsurvey of wholesale and retail seafood products originating fromCanada and other countries. Based on the low recovery ofL. monocytogenes in shrimp and smoked salmon, they concludedthe observed levels did not represent a serious health hazard. In1994, however, a study examining Listeria spp. contamination ofretail RTE fish in Newfoundland found 18.3% (11/60) of cod sampleswere contaminated with L. monocytogenes (Dillon et al., 1994). Incontrast, a report on government seafood testing in 2000 revealedL. monocytogenes contamination in 0.3e0.88% of imported productsand its absence in domestic products (Farber, 2000). Similarly, a lowprevalence of L. monocytogenes in raw and RTE meats from retailwas observed in Alberta (Bohaychuk et al., 2006).

Looking at other Canadian provinces, and particularly B.C.,limited data exist on the occurrence of Listeria spp. in RTE productsand the associated risks of listeriosis linked to consumption ofcontaminated RTE foods. A recent survey of food-processing facil-ities reported inadequate sanitation and food safety practices ina number of B.C.’s fish processing facilities, which lead to L. mono-cytogenes-contamination of a variety of RTE fish products across theprovince (Kova�cevi�c et al., 2012). The survey, however, did not lookat the contamination levels at retail. From this study, a number ofconcerns were raised about the nature of RTE fish productssold in bulk, their handling at retail and high potential forcross-contamination, and the subsequent difficulties in tracingand recalls of such products contaminated with L. monocytogenes(Kova�cevi�c et al., 2012).

Regardless of the size, retail establishments that sell foods withinB.C. are not required to test products or food handling areas forListeria spp. However, these establishments are inspected at leastonce per year and their foods tested as part of the provincial FoodQuality Check Sampling Program (BCCDC, 2010). This program isprimarily educational, with bacteriological tests (e.g. indicatororganisms) used as sanitation checks to inform inspectors andproducers of the effectiveness of current food handling practices,but does not include testing for Listeria spp. or other foodbornepathogens. Foods produced within B.C. for retail outside of provinceare inspected by the federally regulating Canadian Food InspectionAgency (CFIA), and are subject to more intensive microbiologicalmonitoring programs, including testing for Listeria spp. (CFIA, 2011).

Generally, food testing for Listeria spp. occurs at the food-processing level, providing little information on the microbialquality and safety of food at retail. This is a concern becauseL. monocytogenes populations can increase during shipping andprolonged storage, particularly if RTE foods are stored at temper-atures above 4 �C (Farber et al., 2000; Glass and Doyle, 1989).Additional handling of RTE foods at the retail, such as slicing,weighing, and packaging, may increase the potential for cross-contamination (Lin et al., 2006). The current Canadian policy onListeria allows 100 CFU/g of L. monocytogenes in RTE foods in whichproliferation of the organism to levels above this before the endof the product’s shelf-life is not possible. However, extensivemicrobial challenge of retail products is required to determine lis-terial growth potential. Failure to control the proliferation ofL. monocytogenes using extrinsic and intrinsic mitigation strategiesmay allow for unacceptable levels of the organism in products,particularly towards the end of the shelf-life.

Considering the lack of data on the occurrence of Listeria spp.and L. monocytogenes in RTE foods at the retail level in B.C., and the

inconsistent reports on consumer health risks associated withL. monocytogenes-contamination of RTE foods, the goals of thisstudywere two-fold: (1) test RTEmeat and fish products from retailestablishments for the presence of Listeria spp.; and, (2) investigatepossible health risks associated with recovered L. monocytogenesthrough genotypic and phenotypic characterization.

2. Materials and methods

2.1. Sample collection

Ready-to-eat meat and fish products were purchased from sevenlarge chain retail establishments and 10 smaller retailers in theMetro Vancouver area (B.C., Canada) in September and October2010. Overall, 80 samples were collected: 40 deli meats and 40 RTEfish products. Meat samples included: beer sausage, bologna,cervelat and genoa salami, cheese loaf, chicken and turkey breast,cooked ham, corned beef, meat macaroni loaf, mortadella, varietypack sausages, and different types of pepperoni (e.g. beef, chicken,turkey). Fish samples consisted of different flavoured, candied and/or smoked fish jerky, nuggets, and pepperoni samples, as well aslox, sockeye sticks, smoked steelhead trout, and tuna. Samples(approximately 50 g) were purchased as sliced/weighed deli prod-ucts or in manufacture-sealed packaging. Samples were transportedto the laboratory in coolers on the day of purchase, and tested priorto best before/expiry date.

2.2. Isolation of Listeria spp.

Samples were analyzed according to Health Canada’s MFLP-74enumeration (Pagotto et al., 2002) and MFHPB-30 two-stepenrichment (Pagotto et al., 2001) methods. Confirmation of Listeriaspp. was based on Gram stain, catalase and oxidase reactions, andmotility at room temperature. Isolates were speciated by standardbiotyping (Microgen Listeria ID, Microgen Bioproducts Ltd., Cam-berley, Surrey, U.K. and API Listeria, BioMerieux, Marcy l’Etoile,France).

2.3. Serotyping and genetic fingerprinting

Isolates were serotyped by slide agglutination and antiseraprepared according to Seeliger and Höhne (1979) at the CanadianNational Microbiology Laboratory. Genetic fingerprinting based onpulsed-field gel electrophoresis (PFGE) was performed according toPulseNet standardized protocol at the Canadian Listeriosis Refer-ence Service Laboratory using restriction enzymes AscI and ApaI(Gilmour et al., 2010). PFGE patterns were assigned after compar-ison to the PulseNet Canada database.

2.4. Screening for Listeria genomic island and internalin A profiling

Conventional polymerase chain reactions (PCR) were used toscreen for the presence of the 50 kb Listeria genomic island (LGI1)and amplification of the 2.4 kb inlA gene. Briefly, LGI1 screeningwas performed using two sets of primers (Table 1). DNA was iso-lated from overnight cultures grown on Tryptic Soy Agar (Difco,Becton Dickinson Diagnostics, Mississauga, ON, Canada). A singlecolony was resuspended in 100 ml of 1 � TriseEDTA buffer, heatedat 90 �C for 10 min, cooled on ice for 2 min, and centrifuged(16,000 � g for 5 min). PCR reactions (25 mL) using 5 U of AmpliTaqGold 360 DNA polymerase (Applied Biosystems, Life Technologies,Carlsbad, California, USA), 0.4 mM of respective primers, 200 mMdNTPs (Invitrogen Canada Inc., Burlington, ON), and template DNA(1 mL) were cycled as follows: 95 �C for 5 min; 35 cycles of 94 �C for30 s, 52 �C for 30 s and 72 �C for 20 s; followed by 72 �C for 5 min.

Table 1Oligonucleotide primers used in the study.

Primer (Tm) Sequence (50e30) Product Size

Listeria Genomic IslandLGI1-1861-F (52 �C) GAT ACT GGC GAA AGC TTC TA 316 bpLGI1-1861-R (50 �C) GGT TTC GGG TTA ATG ATG TALGI1-1862-F (53 �C) GAG CAA CAC CAC CTA AGT TC 299 bpLGI1-1862-R (52 �C) CAG TCG CTA TCG TAC TTG AAinlA AmplificationinlA-JK-F (47.6 �C) TAC AAC GAA ACC TGA TAT TG 2496 bpinlA-JK-R (47.5 �C) GCT AGA TAT AGT CCG AAA AC

J. Kova�cevi�c et al. / Food Microbiology 30 (2012) 372e378374

Images of ethidium bromide-stained bands were visualized on1% agarose gel using Image Master VSD (Amersham PharmaciaBiotech, Uppsala, Sweden). L. monocytogenes 08-5578 (Gilmouret al., 2010) strain obtained from the Canadian National Microbi-ology Laboratory was used as a positive control.

Similarly, for inlA amplification, 5 U of AmpliTaq Gold 360 DNApolymerase was used with two sets of custom primers (Table 1),each at 0.5 mM, 200 mM dNTPs (Invitrogen), and DNA template(50e100 ng) obtained using DNeasy Blood and Tissue Kit (Qiagen,Toronto, ON). The thermocycling parameters were: initial dena-turation at 94 �C for 2 min; 20 cycles of 94 �C for 1 min, 60e50 �Cfor 1 minwith touchdown decrease of 0.5 �C per cycle, and 72 �C for2.5 min; 20 cycles of 94 �C for 1 min, 50 �C for 1 min, and 72 �C for2.5 min; final extension at 72 �C for 7 min (Van Stelten et al., 2010).PCR product was purified using QIAquick PCR Purification kit(Qiagen). Sequencing of the inlA PCR product was performed atCanada’s Michael Smith Genome Science Centre using custom(Table 1) and previously published primers (Van Stelten et al.,2010), which amplified six regions of the inlA gene. Nucleotidesequences were assembled and analyzed with Geneious 5.4 soft-ware (Biomatters Ltd., Aukland, New Zealand). The presence ofpremature stop codons (PMSC) was determined by comparingobtained inlA sequence data to that of the L. monocytogenes EGDereference strain (Glaser et al., 2001).

2.5. Antimicrobial resistance screening

Antimicrobial resistance was assessed by disc diffusion assayaccording to CLSI guidelines (Clinical and Laboratory StandardsInstitute, 2010) and a modified Mesak and Davies protocol(Mesak and Davies, 2009). Briefly, bacterial cultures were grown at35 �C for 18 � 2 h in Tryptic Soy Broth (Difco), diluted to1 � 107 CFU/mL in tempered 0.75% agar (45 �C), mixed gently, andimmediately poured onto MullereHinton agar (Difco). Once theoverlay agar solidified, antimicrobial susceptibility test discs (BBL�

Table 2Genotypic and phenotypic properties of Listeria monocytogenes isolates recovered from r

ID-Isolate No. Food Source Retail Establishment Se

LR39-1 Smoked salmon RE7 1/

LR39-2 Smoked salmon RE7 1/

LR39-3 Smoked salmon RE7 1/

LR59-1 Smoked salmon fingers RE11 1/

LR59-2 Smoked salmon fingers RE11 1/

LR59-3 Smoked salmon fingers RE11 1/

a PFGE, Pulsed Field Gel Electrophoresis.b LGI1, Listeria Genomic Island 1; Absent (�), present (þ).c *, indicates reduced susceptibility.

Sensi-Disc�, BD Diagnostics, Sparks, MD, USA) were applied andplates incubated at 35 �C for 24 h. A panel of 18 antimicrobials(AMs; mg) comprising 11 classes of antibiotics were used: amikacin(AMK; 30), ampicillin (AMP; 10), cefoxitin (FOX; 30), chloram-phenicol (CHL; 30), ciprofloxacin (CIP; 5), clindamycin (CLI; 2),erythromycin (ERY; 15), gentamicin (GEN; 10), imipenem (IPM; 10),kanamycin (KAN; 30), linezolid (LZD; 30), nalidixic acid (NAL; 30),rifampin (RIF; 5), streptomycin (STR; 10), trimethoprim with sul-famethoxazole (SXT; 10), tetracycline (TET; 30), trimethoprim(TMP; 5), and vancomycin (VAN; 10). Zones of inhibition weremeasured (mm) at 24 h. Since no resistance criteria exist for Listeriasusceptibility testing in CLSI guidelines for the tested AMs otherthan AMP and SXT, criteria for staphylococci were applied (Clinicaland Laboratory Standards Institute, 2011). Escherichia coli MG1655was used as a quality control strain.

3. Results

3.1. Recovery of Listeria spp. in retail food samples

From 80 RTE food samples analyzed, eight (10%) were positivefor Listeria spp., with all containing less than 100 CFU/g. Positivesamples came from four retail establishments; three small and onelarge establishment. Listeria welshimeri was the most commonlyisolated species (4/8), followed by Listeria innocua (2/8) andL. monocytogenes (2/8). Listerial contamination was seen exclusivelyin RTE fish samples, and included smoked and candied salmon andsalmon jerky.

Two smoked salmon samples contaminated with L. mono-cytogenes were purchased from different retail establishments,and originated from different food-processing facilities; onefacility was federally registered and inspected by the CFIA whilethe other is under provincial inspection authority. L. mono-cytogenes recovered from one smoked salmon sample belongedto serotype 1/2a while the isolates recovered from the secondsample were 1/2b. Correspondingly, PFGE patterns were dissimilarbetween L. monocytogenes isolates from different samples, butwere identical for all L. monocytogenes recovered from respectivepositive samples (Table 2).

3.2. Genotypic properties of L. monocytogenes isolates

Listeria genomic island 1 was detected in three 1/2a L. mono-cytogenes clonal isolates but not in 1/2b isolates or isolates ofL. innocua (n ¼ 2) and L. welshimeri (n ¼ 4) (Fig. 1). DNA sequencingrevealed the absence of mutations resulting in PMSCs within inlAof all L. monocytogenes isolates. However, serotype 1/2a isolates

eady-to-eat salmon in Vancouver, B.C.

rotype PFGEa LGI1b Antimicrobial Resistancec

2a LMACI.0001 þ CIP*, CLI, FOX, NALLMAAI.0001

2a LMACI.0001 þ CIP*, CLI, FOX, NALLMAAI.0001

2a LMACI.0001 þ CIP*, CLI, FOX, NALLMAAI.0001

2b LMACI.0470 � CIP*, CLI, FOX, NALLMAAI.0584

2b LMACI.0470 � AMK, CIP*, CLI, FOX, NAL, STRLMAAI.0584

2b LMACI.0470 � AMK*, CIP*, CLI, FOX, NAL, STRLMAAI.0584

Fig. 1. PCR confirmation of Listeria genomic island 1 (LGI1) presence in L. monocytogenes and other Listeria spp. recovered from ready-to-eat fish samples from retail, using LGI1-1861 primers.

J. Kova�cevi�c et al. / Food Microbiology 30 (2012) 372e378 375

possessed eight non-synonymous and 26 synonymous mutations,while 19 non-synonymous and 63 synonymous mutations wereobserved in the 1/2b isolates (Fig. 2).

3.3. Antimicrobial resistance of Listeria spp.

All L. monocytogenes isolates were resistant to CLI, FOX andNAL, and had reduced susceptibility to CIP (Table 2). Similarly,L. welshimeri isolates were resistant to CLI, FOX and NAL (Table 3).Three L. welshimeri isolates had reduced susceptibility to CIP,while two exhibited reduced susceptibility to CHL. L. innocua wereresistant to FOX and NAL, and had reduced susceptibility to CIP andCLI (Table 3). Two of the three clonal 1/2b L. monocytogenes isolateswere resistant to STR; one was also resistant to AMK while theother exhibited reduced susceptibility (Table 2).

4. Discussion

The results of the current study show that the presence ofListeria spp. in retail RTE foods in B.C. is not uncommon. Evenwith a limited number of samples tested, Listeria spp., including

Fig. 2. Full-length internalin A illustration, with the scale below representing amino-L. monocytogenes strains from ready-to-eat fish samples (B).

L. monocytogenes, were recovered from RTE foods, specifically,smoked fish samples. The data correspond with a recent study thatinvestigated the occurrence and distribution of Listeria spp. in food-processing facilities across the B.C. province (Kova�cevi�c et al., 2012).Similar to the findings reported here, the authors did not recoverL. monocytogenes from RTE meat products (Kova�cevi�c et al., 2012).Also, contamination levels of fish reported in the 2009 study werecomparable to those seen in the current study. In 2009, 28%of smoked fish samples from food-processing facilities in B.C.were contaminated with Listeria spp., while 20% harbouredL. monocytogenes (Kova�cevi�c et al., 2012). Similar levels of Listeriaspp. in retail fish samples (20%) were observed in the present study,but only 5% were identified as L. monocytogenes.

Outside of Canada, varying levels of listerial contamination infish samples have been reported in coastal European countries(Cabedo et al., 2008; Garrido et al., 2009; Gianfranceschi et al.,2003; Van Coillie et al., 2004). A 2009 study in Spain found Lis-teria spp. in 18.6% of smoked salmon samples from retail, with 10%harbouring L. monocytogenes (Garrido et al., 2009). A Spanish studyin 2008 (Cabedo et al., 2008) recovered L. monocytogenes from 7.9%of smoked salmon samples, which is comparable to levels observed

acid position (A), and the respective region-specific polymorphisms observed in

Table 3Genotypic and phenotypic properties of Listeria isolates other than L. monocytogenes.

ID-Isolate No. Species Retail Establishment Food Source LGI1a Antimicrobial Resistanceb

LR43-1 L. welshimeri RE7 Smoked salmon � CHL*, CIP*, CLI, FOX, NALLR58-1 L. welshimeri RE11 Wild smoked lox trim � CHL*, CIP*, CLI, FOX, NALLR61-1 L. welshimeri RE11 Maple smoked salmon nuggets � CHL*, CLI, FOX, NALLR64-1 L. welshimeri RE12 Smoked salmon jerky � CIP*, CLI, FOX, NALLR67-1 L. innocua RE13 Salmon candy � CIP*, CLI*, FOX, NALLR69-1 L. innocua RE13 Salmon strips � CIP*, CLI*, FOX, NAL

a LGI1, Listeria Genomic Island 1; Absent (�), present (þ).b *, indicates reduced susceptibility.

J. Kova�cevi�c et al. / Food Microbiology 30 (2012) 372e378376

in the current study. In contrast, in Italy (Gianfranceschi et al., 2003)and Belgium (Van Coillie et al., 2004) much higher levels ofL. monocytogenes were reported in fish and fish products (27.9%)and smoked halibut (33.3%), respectively. However, it is importantto note that these studies involved a large number of samples,and in some cases a longitudinal approach, limiting the extent ofcomparison between studies.

Generally, risk posed to consumers in foods contaminatedwith L. monocytogenes increases with higher concentrations of theorganism (EFSA, 2007; FAO and WHO, 2004). Recently, however, ithas been recognized that different strains of L. monocytogenes maybe associated with increased virulence potential (Ondrusch andKreft, 2011; Orsi et al., 2010; Schmid et al., 2009). Highly virulentL. monocytogenes strains may cause disease at relatively lowmicrobial concentrations (Graves et al., 2005). Further, evidencerelating to novel genetic properties contributing to listeriosiscontinues to be reported (Bundrant et al., 2011; Gilmour et al., 2010;Graves et al., 2005). In the current study, both L. monocytogenes-positive samples had less than 100 CFU/g. However, one strain(LR39; Table 2) possesses striking similarity to the highly virulentstrains responsible for the 2008 Canadian listeriosis outbreak(Gilmour et al., 2010). In addition to being serotype 1/2a and havinga matching PFGE pattern (Table 2) to that of L. monocytogenes 08-5923 isolate from the 2008 outbreak, LR39 isolates also possessedthe 50 kb LGI1 genomic island. To date, this island has only beenreported in L. monocytogenes isolates responsible for the 2008Canadian listeriosis outbreak. The exact role of this island has notbeen elucidated, though its encoded genetic complement, whichincludes putative type II and type IV secretion systems, pilus-likesurface appendages, a multidrug efflux pump homologue (EmrE),and an alternate sigma factor, suggests it may play a role in bacterialpersistence and/or pathogenicity (Gilmour et al., 2010).

Additional genotypic properties, such as an inlA gene coding forthe full 800-amino acid virulent form of the InlA surface protein,suggest that L. monocytogenes isolates recovered from fish samplesin the present study pose risk to B.C. consumers. The role of InlA inbacterial invasion has beenwell established (Milillo andWiedmann,2009; Orsi et al., 2007). Mutations in inlA result in the expression ofa truncated form of the protein which is incapable of effectivelyanchoring to the bacterial cell wall (Jonquieres et al., 1998;Nightingale et al., 2005). As a result, bacteria exhibit reduced abilityto invade Caco-2 cells and subsequently cause listeriosis in animalmodels (Nightingale et al., 2005, 2008). The absence of prematurestop codons in the inlA gene in L. monocytogenes strains reportedhere shows the isolates are capable of producing full-length InlArequired for host cell invasion. Interestingly, some non-synonymousmutations were observed in inlA gene of LR39 strain whencompared to the EGDe reference strain, but were identical to thosereported for the 2008 Canadian outbreak strains (Gilmour et al.,2010). The number of point mutations in the inlA gene of LR59strain was more than double for non-synonymous and almost threetimes higher for synonymous mutations compared to LR39 strain(Fig. 2). In accordancewith other published reports (Orsi et al., 2007;

Van Stelten and Nightingale, 2008), mutations were primarily seenin B repeats and membrane anchor regions, with a minorityalso occurring in the leucine rich repeats (LRR) region (Fig. 2). TheLRR domain promotes interaction with human surface receptor,E-cadherin, and has been reported as highly conserved, especiallyfrom repeats 7 to 15 (Bierne et al., 2007; Schubert et al., 2002). Incontrast to previously reported low level of mutations in the highlyconserved intergenic region (Schubert et al., 2001), our LR59 strainhad seven non-synonymous and 11 synonymous mutations in thisregion (Fig. 2). The absence of PMSC in the inlA gene of both LR39and LR59 strains, and high similarity to the 2008 outbreak strainssuggest the InlA produced by these strains is capable of promotingeffective invasion of host cells.

In addition to the interesting genotypic properties detailedabove, recovered strains also exhibited unexpected phenotypeswhen screened for antimicrobial resistance. All L. monocytogenesand L. welshimeri strains tested were resistant to CLI, whileL. innocua strains exhibited reduced susceptibility. Previous studiesexamining listerial sensitivity to CLI reported varying degrees ofresistance across species (Chen et al., 2010; Davis and Jackson,2009; Troxler et al., 2000). It has been suggested that listerialresistance to CLI may be due to modification of the 23S ribosomalRNA or the presence of an enzyme capable of modifying the anti-microbial, resulting in its inactivation (Chen et al., 2010; Depardieuet al., 2007). Clindamycin inhibits bacterial protein synthesis bybinding to the 50S subunit of the bacterial ribosome, a similarresistance mechanism to that associated with reduced efficacy ofERY and CHL, which raises concerns about the potential for cross-resistance to differing antibiotic classes through a common mech-anism (Depardieu et al., 2007). While no resistance to ERY wasobserved in recovered isolates, reduced susceptibility to CHL wasseen in three L. welshimeri strains (Table 3).

Interestingly, reduced susceptibility to CIP was observed for allstrains with the exception of one L. welshimeri isolate (LR61-1).This was unexpected as previously published reports looking at CIPactivity against Listeria spp. from clinical, animal, food, and envi-ronmental origins reported low rates of resistance (Davis andJackson, 2009; Safdar and Armstrong, 2003; Troxler et al., 2000).However, the extent of comparison between studies is hamperedby differences in the methods used to determine resistance (e.g.microdilution broth vs. disc diffusion assay; media differences).Further, a lack of consensus on antibiotic concentrations and/orbreakpoints associated with reduced susceptibility and resistancefor Listeria spp. make effective comparison across studies difficult(Troxler et al., 2000).

In general, antimicrobial intervention is required for the treat-ment of invasive listeriosis infections, with clinicians commonlyusing AMP in combination with GEN, or SXT (Hof et al., 1997;Schlech, 2000). In this study, no resistance to commonly employedtherapeutic agents was observed for L. monocytogenes or otherListeria spp. This was expected, as L. monocytogenes isolates aregenerally considered susceptible to most antibiotics, withthe exception of cephalosporins and fosfomycin for which innate

J. Kova�cevi�c et al. / Food Microbiology 30 (2012) 372e378 377

resistance has been described (Hof et al., 1997; Troxler et al., 2000).Accordingly, all strains were resistant to FOX, a second-generationcephalosporin, but not to other b-lactams tested (Tables 2 and 3).This phenomenon has been ascribed to the low binding affinity ofcephalosporins to penicillin-binding proteins (PBPs) present in Lis-teria spp. cytoplasmic membranes (Hof et al., 1997; Troxler et al.,2000). It was unusual, however, to see resistance to AMK andSTR in two clonal isolates of L. monocytogenes 1/2b serotype (LR59-2and 3; Table 2). Amikacin and STR, along with GEN and KAN, belongto the aminoglycoside class of antibiotics, which are employedtherapeutically for listeriosis treatment (Schlech, 2000). Listerialresistance to this class of antimicrobials is rarely reported (Chenet al., 2010; Morvan et al., 2010). Of 4668 clinical L. monocytogenesisolates recently tested in France, only three were resistant to STR;two were resistant to lower concentrations (4e6 mg/mL), while oneexhibited resistance at 256 mg/mL (Morvan et al., 2010). When itcomes to food and environmental strains, none of the 49L. monocytogenes isolates from food and environmental sourcestested in the U.S. in 2009 possessed resistance to STR (1 mg/mL);however, all six L. welshimeri recovered from animal sources wereresistant (Davis and Jackson, 2009). In 2010, another U.S. study re-ported reduced susceptibility to STR (10 mg) in 2% (2/80) ofL. monocytogenes strains recovered from catfish fillets and respectiveprocessing environments (Chen et al., 2010). Recently, however, ithas been reported that sublethal exposure to triclosan, a broad-spectrum biocide incorporated into variety of commercially usedproducts, promotes resistance to various aminoglycosides, includingGEN, KAN, STR, and tobramycin (Christensen et al., 2011). Resistanceto low and high concentrations of the antibiotic has been suggestedto be associated with possible ribosomal mutations and/or theproduction of 6-N-streptomycin adenyltransferase encoded by theaad6 gene (Morvan et al., 2010). These developments are alarming,considering that gentamicin combined with ampicillin, is apreferred choice for listeriosis treatment (Hof et al., 1997).

In summary, the research findings suggest that B.C. consumersof RTE fish products are exposed to L. monocytogenes. Based on thegenetic properties observed in recovered L. monocytogenes isolatesfrom this study, including the presence of LGI1, previously unre-ported in isolates from fish and retail food, as well as PMSC-lackinginlA gene sequences required for the production of InlA proteinand bacterial invasion of the host cell, it is reasonable to assumeL. monocytogenes found in B.C. RTE fish have potential to causelisteriosis, and is concerning. Current work is under-way toevaluate the contribution of LGI1 to food chain-relevantstress tolerance, persistence, and virulence of this organism.Also, despite not observing resistance to first-line antimicrobialagents used to treat invasive listeriosis, resistance and reducedsusceptibility to a number of clinically relevant antimicrobialswas observed and is of concern. Future research investigatingthe antimicrobial resistance, prevalence, and virulence potential ofisolates originating from the B.C. food supply, and in particular fish,is required to further define current risks faced by consumers.

Acknowledgements

We would like to acknowledge Dr. Matthew Gilmour and othercolleagues from the Canadian National Microbiology Laboratory fortheir assistance. We also extend our thanks to Dr. Franco Pagottoand the Public Health Agency of Canada for serotyping and geneticfingerprinting of the isolates.

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