Antimicrobial resistance of Listeria monocytogenes isolated from dairy-based food products

Science of the Total Environment 407 (2009) 4022–4027

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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Antimicrobial resistance of Listeria monocytogenes isolated from dairy-based food products Steve Harakeh a,⁎, Imane Saleh b, Omar Zouhairi a, Elias Baydoun a, Elie Barbour c, Nisreen Alwan d a

Biology Department, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon Dubai, P.O. Box 15495, United Arab Emirates Department of Animal Sciences, American University of Beirut, P.O. Box 11-0236, Beirut, Lebanon d Forschungsinstitut Senckenberg, Sektion Ichthyologie, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany b c

a r t i c l e

i n f o

Article history: Received 5 February 2009 Received revised form 7 April 2009 Accepted 7 April 2009 Keywords: Listeria monocytogenes Dairy-based food products Baladi cheese, Shankleesh, Kishk Antimicrobial resistance

a b s t r a c t In this study Listeria monocytogenes (L. monocytogenes) was isolated from three traditionally consumed Lebanese dairy-based food products. One hundred and sixty four samples (45 samples of Baladi cheese, 36 samples of Shankleesh and 83 of Kishk) were collected from the Bekaa Valley in the Northeast region of Lebanon. Suspected Listeria colonies were selected and initially identified by using standard biochemical tests. Initial identification of the positive L. monocytogenes colonies was confirmed at the molecular level by Polymerase Chain Reaction (n = 30) and the confirmed isolates were evaluated for their susceptibility to 10 commonly used antimicrobials. All of the 30 isolates were confirmed to be L. monocytogenes yielding a PCR product of ∼ 660 base pairs (bp). L. monocytogenes was detected in 26.67%, 13.89% and 7.23% of the Baladi cheese, Shankleesh and Kishk samples, respectively. The highest resistance in L. monocytogenes isolates was noted against oxacillin (93.33%) followed by penicillin (90%). The results provide an indication of the contamination levels of dairy-based foods in Lebanon and highlight the emergence of multi-drug resistant Listeria in the environment. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The importance of dairy-based food as a vehicle for the transmission of various diseases has been documented; especially in countries where hygienic standards are not strictly enforced (Meyer-Broseta et al., 2003). Contaminated milk and its by-products may harbour a variety of microorganisms which are responsible for many food-borne outbreaks (Danielsson-Tham et al., 2004; MacDonald et al., 2005; Makino et al., 2005; Okwumabua et al., 2005; Oliver et al., 2005). Listeria is considered to be one of the most important causes of food-borne diseases. L. monocytogenes, a ubiquitous gram-positive microaerophilic bacterium, is capable of causing severe Listeriosis infections in humans (encephalitis, meningitis and septicaemia especially in immunocompromised individuals) and animals (mastitis, diarrhea and gastroenteritis) (Herman et al., 1995; Vela et al., 2001; Siegman-Igra et al., 2002; McLauchlin et al., 2004; Aygun and Pehlivanlar, 2006). L. monocytogenes has been involved in many outbreaks and sporadic cases of disease primarily associated with the consumption of pasteurized milk, cheeses made from unpasteurized milk and other dairybased products that serve as good medium for the growth and survival of many pathogenic organisms in both industrialized and developing

⁎ Corresponding author. Tel.: +961 3570119; fax: +961 5468476. E-mail address: [email protected] (S. Harakeh). 0048-9697/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2009.04.010

countries (Kells and Gilmour, 2004; Makino et al., 2005; Manfreda et al., 2005). Usually, the presence of any Listeria species in food is an indication of microbial contamination (Gilot and Content, 2002). The identification of Listeria using biochemical standard tests is laborious, time-consuming and an inaccurate procedure. For this reason and for better accuracy, PCR was used, relying on nucleic acid composition of the bacterium rather than the phenotypic expression that might vary in accordance with growth conditions (Cocolin et al., 2002). The gene encoding for the invasive-associated protein (iap) is common to all Listeria species (Gilot and Content, 2002). The gene portions at the 3′and 5′ ends are conserved for all Listeria species whereas the internal portions are species-specific. This characteristic makes the gene an ideal tool for PCR to identify Listeria isolates and to be able to classify them into different species. The iap gene, which encodes for the major extracellular protein p60, is an essential murein hydrolase needed for septum separation in cell division (in a late step). Moreover, p60 contributes to the adherence/attachment of Listeria to certain eukaryotic cells (Bubert et al., 1999). The excessive use of antimicrobials has led to the emergence of antimicrobial-resistant bacteria in the environment. Antimicrobials used as growth promoters in animal feed have reduced the impact of infectious diseases (diarrhea, skin and organ abscesses and mastitis) but led to the dissemination of antimicrobial-resistant L. monocytogenes into the environment (Teuber, 1999; Jansen et al., 2003). Monitoring the antimicrobial resistance of L. monocytogenes in humans and animals is of

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utmost importance in order to (a) perceive changes in the patterns of resistance to commonly used antimicrobials, (b) implement pro-active measures to control the use of antimicrobial agents and (c) prevent the spread of multi-drug resistant strains which can have many undesired consequences (Harakeh et al., 2005). Antimicrobial resistance of L. monocytogenes may be associated with the presence of a plasmid or determined by genes that are transferred by conjugation. Also, mutational events in chromosomal genes can play a role in conferring resistance to Listeria species (Poros-Gluchowska and Markiewicz, 2003). In Lebanon, the Bekaa Valley is an important site for the production of dairy-based food products that are consumed by many on a daily basis. Considering the marked importance of Listeria as food-borne pathogens, this study aims at evaluating the antimicrobial resistance of PCR-confirmed L. monocytogenes organisms isolated from dairy products which are consumed raw in Lebanon. The foods included in this study are: Baladi cheese (Lebanese cheese balls), Shankleesh (a mold ripened cheese) and Kishk (a dried fermented milk–wheat mixture), which are mainly prepared in the Bekaa Valley (Saleh et al., 2009).

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coliforms count, a sample with 104 CFU/g and above is considered unfit for human consumption (Table 1). 2.4. Biochemical identification of the suspected L. monocytogenes isolates Colonies appearing on LSA were first selected based on their morphology then identified by biochemical tests. Black to brown colonies surrounded by black halos were chosen (Hitchins, 1995; Aygun and Pehlivanlar, 2006). Those colonies were Gram stained. Only Gram-positive short rods were further tested for their ability to produce acids from the fermentation of D-xylose and L-rhamnose sugars, and were also subjected to the β-haemolysis test (Cocolin et al., 2002, Zhou and Jiao, 2005). 2.5. DNA extraction

One hundred and sixty four samples of three types of dairy-based foods, Baladi cheese (n = 45), Shankleesh (n = 36) and Kishk (n = 83) were collected from the Bekaa Valley and obtained from various sources (markets, houses and small family dairy farms). Sample collection was done in four field visits from August till December. They were placed in sterile bags, numbered and brought to the laboratory in a small fridge. All samples were refrigerated upon arrival at the laboratory and analysis was carried within 24 h of their arrival (Saleh et al., 2009).

Using a sterile loop, a bacterial colony was suspended in 5 ml of sterile Brain Heart Infusion (BHI) broth (Oxoid, Hampshire, UK). This suspension was incubated using a shaking water bath at 37 °C. DNA extraction was conducted using 1 ml of the bacterial suspension to extract total genomic DNA according to the GFX genomic blood DNA purification kit from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK) (Harakeh et al., 2006). However, the protocol was adjusted to extract the DNA of Gram-positive bacteria. The pellet was lysed with 40 µl lysozyme buffer (0.1 M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA and 5% Triton X-100) instead of proteinase K buffer (12 mM Tris–HCl pH 8.0, 6 mM EDTA and 0.6% SDS) used for Gram-negative bacteria followed by the addition of 10 µl of lysozyme (10 mg/ml in 10 mM Tris–HCl, pH 8.0) instead of proteinase K (20 mg/ml in 10 mM Tris–HCl pH 8.0). DNA concentrations and purity were measured by calculating the ratio of absorbance at 260 nm and 280 nm using a spectrophotometer (Thermo, England). A 100–200 ng/µl DNA template with a 1.8 ratio was used per 25 µl PCR reaction (Harakeh et al., 2005).

2.2. Bacterial isolation and counting

2.6. PCR assays

A 25 g portion of each sample was weighed aseptically into a sterile stomacher bag (Seward Medical Stomacher Bags© Seward, Germany) containing 225 ml of sterilized 1% (w/v) peptone water (HiMedia Laboratories Limited, India) and macerated in a laboratory blender stomacher 400 (Seward, England) for 3 min (Peng and Shelef, 2000). A 10-fold serial dilution, ranging from 10− 1 to 10− 3, was prepared. A selective medium: Listeria Selective Agar (LSA) (Oxoid, Hampshire, UK) containing Listeria selective supplement (Oxford modified) (Oxoid, Hampshire, UK) was used for the isolation, enrichment and plating of Listeria (Gulmez and Guven, 2003). Bacteriological analyses were performed, by plating in duplicates (a volume of 0.1 ml of each dilution on agar plates containing appropriate selective media) (Gulmez and Guven, 2003). All analyses were conducted under aseptic conditions. Plated cultures were then incubated at 35 °C for 48 h (Gulmez and Guven, 2003). Colonies that exhibited the L. monocytogenes morphology were preserved for further analyses. All bacteriological analyses were done according to the Compendium of Methods for the Microbiological Examination of Foods (Downes and Ito, 2001 and Horwitz, 2000).

Molecular characterization and differentiation of the suspected L. monocytogenes colonies, previously confirmed by biochemical tests, were done using standard PCR. The iap gene, which is common to all Listeria species was the specific target (Bubert et al., 1999). In all the PCR experiments, the forward primer List1B [5′-TTATACGCGACCGAAGCCAAC-3′] was derived from the conserved 3′ end of the iap gene which is specific to all Listeria species, whereas the 5′ reverse primer (MonoA) [5′-CAAACTGCTAACACAGCTACT-3′] was specific for L. monocytogenes (Bubert et al., 1999). Amplification of bacterial DNA was performed in 25 µl reaction mixture containing: 1.25 µl of each primer (10 pmol/µl), 3 µl of 50 ng/µl of the purified DNA template, 2.5 µl of 10x PCR reaction buffer (AB-gene products, UK), 1.875 µl of MgCl2 (25 mM) (AB-gene products), 0.5 µl of each dNTP (100 mM) (dATP, dGTP, dCTP and dTTP) (AB-gene products, UK) and 0.2 µl of 5 U/µl of Thermus aquaticus (Taq) DNA polymerase (AB-gene products, UK). The volume was brought up to 25 µl by adding sterile double distilled water. The mixture was placed in a thermal cycler, icycler (Bio-Rad, USA). Negative controls (no DNA template) and

2.3. Identification of the microbiological quality of dairy-based foods

Table 1 Bacteriological quality of dairy foods as determined by CFU ranges according to PHLS.

2. Materials and methods 2.1. Sample collection

According to the guidelines set by the Public Health Laboratory Service (PHLS) (Gilbert et al., 2000), the microbiological quality of ready-to-eat (RTE) foods including dairy products can be grouped into four different categories based on their bacterial counts: (1) Satisfactory, (2) Acceptable, (3) Unsatisfactory and (4) Unacceptable/Potentially hazardous. A sample with Listeria counts of more than or equal to 102 CFU/g is considered unfit for human consumption. As for the total

Microbiological quality (CFU/g) Bacterial type b

APC Total coliforms L. monocytogenes a

Satisfactory

Acceptable

Unsatisfactory

Unacceptable/(PHa)

N/A b 20 b 20

N/A 20–104 20–102

N/A N 104 N 102

N/A N/A N/A

Potentially Hazardous. Aerobic Plate Count.

b

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Table 2 Percentages of samples contaminated with aerobic bacteria and total coliforms (Saleh et al., 2009). Bacterial type

Kishk

Shankleesh

Baladi cheese

Aerobic bacteria Total coliforms

100 14.5

100 38.8

100 82.3

positive controls of L. monocytogenes DNA templates were included in every PCR assay. The cycles used for the amplification of the targeted iap gene are in accordance with those used by Bubert et al. (1999). 2.7. Gel electrophoresis Ten-µl aliquot of each of the PCR products was mixed with 2 µl 6x loading dye (Bio-Rad, USA). Electrophoresis was carried out in 1x TAE buffer (0.04 M Tris–acetate and 0.001 M EDTA) at 90 v for 90 min. An EZ load 100 bp ruler (Bio-Rad, USA) was used as a DNA ladder. PCR products were run on 1% agarose gel containing 0.25 µg/ml ethidium bromide. After the run was completed, the bands were visualized through UV transmission and photographed. 2.8. Antimicrobial susceptibility testing PCR-confirmed L. monocytogenes colonies were tested for their susceptibility to different antimicrobials, using the disk diffusion method as set by the Clinical and Laboratory Standards Institute (CLSI) (Matthew, 2008). Briefly, organisms were grown in BHI broth using a shaking water bath at 37 °C until their turbidity matched that of the 0.5 McFarland turbidity standard (Harakeh et al., 2005). A 0.1 ml of the culture was then inoculated on Mueller Hinton agar plates (Oxoid, England) supplemented with 5% (v/v) sheep blood (Facinelli et al., 1993, Marco et al., 2000; Mayrhofer et al., 2004). Antimicrobial impregnated disks (Bio Merieux, France) were then added. Isolates were tested for their susceptibility to: penicillin (10 U/IE), trimethoprim-sulfamethoxazole (1.25 + 23.75 µg), tetracycline (30 µg), gentamicin (10 µg), erythromycin (15 µg), chloramphenicol (30 µg), clindamycin (2 µg), oxacillin (1 µg), ampicillin (10 µg) and vancomycin (30 µg) (Abuin et al., 1994; Marco et al., 2000; Walsh et al., 2001; Prazak et al., 2002; Schlegelova et al., 2002). To determine the extent of antimicrobial susceptibility, the diameter of the inhibition zone around each antimicrobial impregnated disk was measured to the nearest millimetre after an incubation period of 24–48 h at 37 °C. Using CLSI guidelines, each organism was classified as either resistant (no inhibition of growth), intermediate (partial inhibition of growth) or susceptible (inhibition of growth) to the antimicrobials. Intermediate-resistant and resistant isolates were grouped together (Wayne, 2006).

3. Results 3.1. Bacteriological counts and microbiological quality of the tested dairy products Aerobic plate count (APC) and total coliforms counts of the 164 samples used in this study were published in a previous study by our group as summarised in Table 2 (Saleh et al., 2009). All samples tested positive for aerobic bacteria (100%). The levels of contamination with total coliforms was significantly highest in cheese, followed by Shankleesh and Kishk (⁎P b 0.05). No previous guidelines were set to evaluate the microbiological quality of food based on their APC levels. However, based on the total coliforms counts, and according to the guidelines set by the Public Health Laboratory Service (PHLS), 60% of cheese samples, 25.7% of Shankleesh samples and 3.6% of Kishk samples were unfit for human consumption. As for the Listeria counts, all colonies that grew on LSA with L. monocytogenes suspected morphology were counted. The levels of contamination with Listeria was significantly the highest in cheese, followed by Shankleesh and Kishk (⁎P b 0.05). According to the PHLS guidelines, 35.56% of Baladi cheese samples, 19.44% of Shankleesh samples and 15.66% of Kishk samples were considered unsatisfactory based on their level of contamination with Listeria. Percentages of samples hazardous for human were different, based on the bacterial type used as criteria. More Baladi cheese and Shankleesh samples were unfit for human consumption based on their total coliforms level, while more kishk samples were unfit based on their Listeria counts (Fig. 1). 3.2. Identification of Listeria species using conventional biochemical tests Based on their morphology, a total of 107 suspected colonies were selected and subjected to various biochemical tests for further identification. All of the 107 colonies were Gram stained and confirmed to be Gram-positive rods which is a characteristic of Listeria. These were then tested for their ability to ferment xylose. Out of the 107 colonies, 30 colonies tested negative while 77 colonies tested positive. Since all L. monocytogenes biotypes are xylose negative, only the 30 suspected colonies were further tested for their ability to ferment rhamnose. All the suspected colonies showed a yellow colour indicating acid production without the production of gas which is another characteristic of the common biotypes of L. monocytogenes. Colonies were then subjected to β-haemolysis test and were grown on blood agar. The results indicated that all of them showed either complete or partial haemolysis. Therefore, 30 colonies were strongly suspected to be L. monocytogenes, thus they were further confirmed by PCR. 3.3. Molecular characterization of L. monocytogenes using PCR All the suspected L. monocytogenes were confirmed by yielding a PCR product of ∼660 bp using the primer pair MonoA+List1B (Fig. 2).

2.9. Statistical analyses Statistical analyses were carried out using SPSS Version 11.0. Kurtosis showed non-normality within data distribution of the CFU counts and Bartlett's test (for comparison of variances) demonstrated that there were statistically significant differences among standard deviations. This violates one of the important assumptions underlying the analysis of variance (ANOVA) and invalidates most of the standard statistical tests. In such cases, Kruskal–Wallis test based on median comparison was used to compare bacterial counts in different foods, communes, and collection dates. To check if the differences in the percentages of L. monocytogenes contamination levels among the three food types were significant, a χ2 goodness-of-fit test was performed. Finally, Friedman test followed by Nemenyi test were performed to check the significance of the variation in the percentages of antimicrobial resistance levels among different antimicrobials.

Fig. 1. Percentages of Baladi cheese, Shankleesh and Kishk samples unfit for human consumption due to their L. monocytogenes and total coliforms contamination level.

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Fig. 2. L. monocytogenes specific PCR products with the primer pairs MonoA+ List1B. 1: DNA ladder (EZ load 100 bp ruler); 2: negative control.; 3: L. monocytogenes positive strain (obtained from the American Type Culture Collection via the American University of Beirut Hospital, Lebanon); 4–33: DNA amplicons of suspected L. monocytogenes colonies among which 17 were isolated from cheese samples (Lanes: 4–20); 6 from Shankleesh (Lanes: 21–26) and 7 from Kishk samples (Lanes: 27–33).

L. monocytogenes was present in 12 out of 45 cheese samples (26.67%), 5 out of 36 (13.89%) Shankleesh samples and 6 out of 83 (7.23%) Kishk samples.

3.4. Antimicrobial susceptibility of L. monocytogenes isolates Every L. monocytogenes tested isolate was found resistant to at least one of the antimicrobials, with resistance to some of them significantly less common than resistance to the others (⁎P b 0.05). Significantly more isolates were resistant to oxacillin (93.33%), penicillin (90%) and ampicillin (60%) than to the rest of the evaluated antimicrobials (⁎P b 0.05). L. monocytogenes isolates showed relatively high susceptibility to gentamicin (93.34%), trimethoprim-sulfamethoxazole (83.33%), tetracycline (80%), and erythromycin (73.34%) (Fig. 3). Surprisingly, 26.66% of our L. monocytogenes isolates were vancomycin resistant. Although, it is not one of the highest levels of resistance encountered in the study, the result is still alarming considering vancomycin as one of the last therapeutic options to treat human infections.

Fig. 3. The percentage of antimicrobial resistance patterns of the 30 L. monocytogenes isolates. The antimicrobial codes are: P: Penicillin; SXT: Trimethoprim-sulfamethoxazole; TE: Tetracycline; GM: Gentamicin; E: Erythromycin; C: Chloramphenicol; CC: Clindamycin; OX: Oxacillin; VA:Vancomycin; AMP: Ampicillin.

4. Discussion In the past few years, there has been an increase in the number of food-borne illnesses in Lebanon. For instance, 657 cases were attributed to food-borne diseases in Lebanon in 2005 (ESUMOPH, 2005). This has emphasized the need for implementing control measures by the food industry to ensure the wholesomeness of foods produced. Considering the discernible importance of L. monocytogenes as an important food-borne pathogen, this study was undertaken to assess the microbiological quality and contamination status of Baladi cheese, Shankleesh and Kishk by this microorganism. Most of the collected samples were either prepared at homes or produced in factories that do not follow modern hygienic practices and in most of the time using unpasteurized milk. The sources of L. monocytogenes in such products may be faecal and environmental contamination during milking, storage and transport, infected cows in dairy farms, poor silage quality and improper handling of these products at the points of sale (Sanaa et al., 1993; Van Kessel et al., 2004). Lack of hygienic practices during the processing and production of these dairy-based products may also contribute to high levels of contamination. For example, cross-contamination may occur after heat treatment or while cheese ripening. The results obtained in a previous study showed that all the samples tested positive for aerobic plate counts which are indicators of aerobic bacteria (Saleh et al., 2009). This finding may reflect the absence of strict hygienic practices in the preparation of those products. A high aerobic plate count level, in general, is indicative of the possible presence of harmful microorganisms and makes the food unsatisfactory for human consumption (Gilbert et al., 2000; Gillespie and Little, 2000). In addition, the presence of total coliforms is an indicator of faecal as well as postprocessing contamination and is reflective of unsanitary conditions practiced during the different stages of food production (Van Kessel et al., 2004). The assumption is that there is an association between the detection of faecal coliforms and pathogenic organisms (Van Kessel et al., 2004). In addition to the previous indicator bacteria, analysis of L. monocytogenes counts was undertaken to examine the safety of dairy products and to determine the possibility of any potential health hazard. The Lebanese Standard Institution (Libnor) is responsible for setting standards to assess the microbiological quality of foods consumed in

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Lebanon. According to Libnor standards, all kinds of cheeses and Kishk sold in the Lebanese markets should be free of Listeria. There was no difference between the Libnor guidelines and PHLS guidelines in terms of percentages of samples unfit for consumption in our study. However a difference in the microbiological quality of the three dairy-based food products was obvious and it could be attributed to differences in their physical and chemical composition. Cheese, in general, showed the highest degree of contamination where L. monocytogenes was able to grow without any barrier such as the high moisture content, low pH values of up to 4.4 and high salt concentration of up to 20% (Bottarelli et al., 1999; Manfreda et al., 2005; Millet et al., 2006). However, Shankleesh had the lowest degree of contamination because it is coated with thyme which has inhibitory effects against bacteria (Toufieili et al., 1995). Also, storing Shankleesh in olive oil provides an anaerobic environment that, only, favours the growth of certain anaerobic bacteria and suppresses the growth of aerobic ones (Toufieili et al.,1995). Kishk is relatively safe and stable at room temperature for a long period of time. This is because of a variety of factors such as its acidic nature (pH almost 3.8), its low moisture content (b10%), its high salt level (2.8 g/100 g NaCl in dried product) and the presence of a wide range of organic acids in it (Tamime and McNulty, 1999). Interestingly, more Kishk samples were unfit for human consumption due to their Listeria contamination level than to their total coliforms contamination level, which indicates that Listeria species are more tolerant to the difficult growth conditions than coliforms. Comparing the data obtained on L. monocytogenes to those published in Japan, a higher contamination with L. monocytogenes was found in 15 out of 19 cheese samples and 11 out of 64 samples collected from cheese manufacturing plants (cow barns, bulk cooler, drainage, faecal samples from workers and production rooms) (Makino et al., 2005). The presence of L. monocytogenes in cheese was also reported in studies from Europe. The results of a study conducted in Italy on 1656 samples of Gorgonzola cheese indicated that L. monocytogenes was detected in cheese samples monitored after packaging (2.1%) and at the end of shelf life (4.1%) (Manfreda et al., 2005). The presence of L. monocytogenes was also of major concern in America. A study conducted in Brazil revealed the presence of L. monocytogenes in 16.7% of each of the Minas Frescal cheese and raw milk (Silva et al., 2003). Many studies were conducted on the presence of L. monocytogenes in pasteurized whole milk and Mexican-style soft cheese since L. monocytogenes has been responsible for many outbreaks in the U.S.A. (McLauchlin et al., 2004). The levels of contamination detected in our study were much higher than those previously reported. This shows the urgent need of implementing hygienic standards in the food industry. On the other hand, high levels of antimicrobial resistance were encountered in the study. The high percentages of resistance in L. monocytogenes to oxacillin, penicillin, ampicillin and chloramphenicol may be attributed to their frequent use in the treatment of various human diseases in Lebanon. Resistance of L. monocytogenes to penicillin and clindamycin may be caused by the excessive use of both drugs in veterinary medicine. Furthermore, resistance to tetracycline may develop as a result of using this drug as a food supplement in animal feed and as a drug of second choice in the treatment of human diseases and resistance to this drug has been previously reported in environmental isolates (Prazak et al., 2002; Walsh et al., 2001). The high susceptibility of isolates to gentamicin and trimethoprim-sulfamethoxazole was evident in this study and it may be due to the fact that these antimicrobials are not used anymore as either therapeutic antimicrobials in veterinary medicine or as growth promoters in conventional animal fattening (Klein et al., 1998). Naturally, most Gram-positive bacteria are susceptible to glycopeptide antibiotics (vancomycin and teicoplanin). Resistance to these compounds is an intrinsic property of some occasional human pathogens (Marco et al., 1998). Unexpectedly, 26.66% of our isolates showed resistance to vancomycin. Such a level of resistance is

extremely high when compared to previous studies conducted around the world. Aureli et al. (2003) in their study on 148 strains of L. monocytogenes had found that all isolates were vancomycin susceptible. On the other hand, only 0.8% of the 120 L. monocytogenes strains studied by Conter et al. (2009) showed resistance against vancomycin. The significance of vancomycin in human infection treatment makes these findings of crucial importance. Further investigations are highly required to study the mechanism behind this level of resistance. The high antimicrobial resistance of L. monocytogenes observed in this study may be caused by the indiscriminate use of antimicrobial agents by the Lebanese population at large and their excessive use in farms to control diseases (Hinton et al., 1986; Klein et al., 1998; Bower and Dueschel, 1999; Geornaras and Holy, 2001). Vancomycin resistant bacteria, which are of major medical concern, have emerged in the first place due to the misuse of vancomycin to treat Gram-positive bacteria (Geornaras and Holy, 2001). Such a problem can have devastating effects on the efficacy of antimicrobials in treating human food-borne diseases. L. monocytogenes isolates may acquire resistance by the acquisition of mobile genetic components such as mobilizable plasmids and conjugative transposons. VanA gene is one of the genes required in case of vancomycin resistance. This gene can be also carried by a large plasmid (Poyart-Salmeron et al., 1990; Charpentier et al., 1995; Marco et al., 1998; Charpentier et al., 1999). Also, mutational events in chromosomal genes can play a role in conferring resistance to Listeria species. The results of this study provide an important baseline for the contamination status of the Lebanese dairy-based foods with L. monocytogenes and preliminary patterns of its resistance to commonly used antimicrobials. The presence of antimicrobial-resistant strains is alarming and constitutes a serious danger to the public health. It is obvious that the increase of the awareness to the importance of controlled use of antimicrobials is crucial to limit the emergence of drug-resistant bacteria. Additional research is definitely recommended to better understand the mechanisms behind bacterial resistance to antimicrobials. References Abuin C, Fernandez E, Sampayo C, Otero J, Rodriguez L, Saez A. Susceptibilities of Listeria species isolated from food to nine antimicrobial agents. Antimicrob Agents Chemother 1994;38:1655–7. Aureli P, Ferrini AM, Mannoni V, Hodzic S, Wedell-Weergaard C, Oliva B. Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics. Int J Food Microbiol 2003;83(3):325–30. Aygun P, Pehlivanlar S. Listeria spp. in the raw milk and dairy products in Antakya, Turkey. Food Control 2006;17:599–682. Bottarelli A, Bonardi S, Bentley S. Presence of Listeria spp. in short-ripened cheeses. Ann Fac Vet Med Parma Univ 1999;19:293–6. Bower CK, Dueschel MA. Resistance responses of microorganisms in food environments. Int J Food Microbiol 1999;50:33–44. Bubert A, Hein I, Rauch M, Lehner A, Yoon B, Goebel W, et al. Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR. Appl Environ Microbiol 1999;65:4688–92. Charpentier E, Gerbaud G, Courvalin P. Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among Gram-positive and Gram-negative bacteria. J Bacteriol 1999;181:3368–74. Charpentier E, Gerbaud G, Jachquet C, Rocourt J, Courvalin P. Incidence of antibiotic resistance in Listeria species. J Infect Dis 1995;172:277–81. Cocolin L, Rantsiou K, Iacumin L, Cantoni C, Comi G. Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods. Appl Environ Microbiol 2002;68:6273–82. Conter M, Paludi D, Zanardi E, Ghidini S, Vergara A, Ianieri A. Characterization of antimicrobial resistance of foodborne Listeria monocytogenes. Int J Food Microbiol 2009;128(3):497–500. Danielsson-Tham ML, Eriksson E, Helmersson S, Leffler M, Ludtke L, Steen M, et al. Causes behind a human cheese-borne outbreak of gastrointestinal listeriosis. Foodborne Pathog Dis 2004;1:153–9. Downes FP, Ito K, editors. Compendium of methods for the microbiological examination of foods,. 4th Edn. Washington, DC: American Public Health Association; 2001. ESUMOPH. Epidemiology surveillance unit of the Ministry of Public Health; 2005. bhttp:// www.public-health.gov.lbN.

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