Phenotypic and Genotypic Characteristics of <i>Listeria monocytogenes</i> Isolated From Dairy and Meat Products

Abbas Bahador; Behrooz Sadeghi Kalani; Firuzeh Valian; Gholamreza Irajian; Lida Lotfollahi

doi:10.17795/ajcmi-26905

Phenotypic and Genotypic Characteristics of Listeria monocytogenes Isolated From Dairy and Meat Products

Abbas Bahador ² , Behrooz Sadeghi Kalani ¹ , Firuzeh Valian ³ , Gholamreza Irajian ^{1 , *} and Lida Lotfollahi ^{4 , *}

Authors Information

¹ Department of Microbiology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, IR Iran

² Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, IR Iran

³ Department of Microbiology, Faculty of Medicine, Ilam University of Medical Sciences, Ilam, IR Iran

⁴ Department of Microbiology, Faculty of Medicine, UrmiaUniversity of Medical Sciences, Urmia, IR Iran

Corresponding Authors:

Article Notes and Dates

Avicenna Journal of Clinical Microbiology and Infection: August 2015, 2 (3); e26905.Published Online: August 1, 2015

Article Type: Research Article; Received : January 11, 2015; Revised : March 11, 2015; Accepted : April 15, 2015
DOI: http://dx.doi.org/10.17795/ajcmi-26905

To Cite : Bahador A, Sadeghi Kalani B, Valian F, Irajian G, Lotfollahi L. et al. Phenotypic and Genotypic Characteristics of Listeria monocytogenes Isolated From Dairy and Meat Products, Avicenna J Clin Microb Infec. 2015 ;2(3):e26905. doi: 10.17795/ajcmi-26905.

Abstract

Copyright: Copyright © 0, Avicenna Journal of Clinical Microbiology and Infection. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

1. Background

Listeria monocytogenes is an omnipresent bacterium that can cause severe disease in humans and animals (1). For instance, although there were only 2500 ailments each year in the US, L. monocytogenes infections account for 4% of all hospitalizations and 28% of all deaths from foodborne illnesses (2). According to food net US, listeriosis is responsible for 30% of foodborne deaths from 1996 to 2005 and had a high case fatality rate of 16.9% (3). The main mode of transmission of Listeria is through environmental impurity of ready-to-eat food products throughout their production and processing (4-6). In recent years, several outbreaks of listeriosis have been related to contaminated trading nutrients, for instance vegetables, milk, and meat foodstuffs (7, 8).

Listeria infection typically needs antimicrobial treatment to heal. Recommendations are penicillin G or ampicillin combined (or not) with an aminoglycoside. However, little information is available on the resistance of L. monocytogenes, predominantly for non-human-origin isolates (9). Isolates of L. monocytogenes should be characterized in order to precisely diagnose their infection, comprehend the epidemiology of infection, consider outbreaks, and efficiently prevent and reduce the spread of Listeria through the food chain and other routes. Since many strains of L. monocytogenes exist, it is indispensable to have a sound system of subtyping so that the most effective strategies can be implemented for the control of outbreaks (10). The phenotypic subtyping methods are usually less sensitive, have low distinctive ability, and are not easy to reproduce, whereas the genotypic methods are more sensitive and reliable (10). In recent years, multiple Locus VNTR analysis (MLVA) is used for subtyping L. monocytogenes strains (11). “VNTR” is a “variable-number tandem repeat”. MLVA is a method employed for the genetic analysis of particular microorganisms such as pathogenic bacteria. It takes advantage of the polymorphism of tandemly repeated DNA sequences and utilizes the naturally occurring variation in the number of tandem repeated DNA sequences found in many different loci in the genome of a variety of organisms. This method is widely used to assess the molecular fingerprint of microorganisms such as bacteria and perform molecular typing of particular bacteria. There are several methods for L. monocytogenes typing, including repetitive element palindromic PCR (rep-PCR), multilocus sequence typing (MLST), esterase electrophoresis (zymotyping), random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and restriction fragment length polymorphism (RFLP) of ribosomal DNA (ribotyping), however, MLVA method has a high discriminatory power with high-throughput screening that is fairly inexpensive, easy to perform, rapid, and reliable. Therefore, MLVA technique is well suited to interlaboratory comparisons during epidemiological investigations of infection and preferentially it is used in molecular typing of bacteria. According to previous studies, MLVA has a sensitivity equal to that of PFGE and a specificity superior to that of PFGE (12-15).

2. Objectives

3. Materials and Methods

3.1. Bacterial Strains

During November 2010 to December 2013, a total of 317 samples (including dairy products and meat processed) were collected from Tehran, Iran (Table 1). All samples were transferred to TSBYE (Tryptic soy broth positive 0.6% yeast extract, Merck, Germany) and incubated at 4°C. After 7 - 16 days till 6 months incubation, samples were cultured on PALKAM Agar (Merck, Germany) and Listeria selective agar (Himedia, India), then, plates were incubated at 35°C for 24-48 h. Grown colonies were recognized using microbiological and biochemical tests, including Gram staining, catalase reaction, oxidase test, hemolysis on Sheep Blood Agar, Christie Atkins Munch Petersen (CAMP) test, Voges-Proskauer (MR-VP), Methyl Red tests and fermentation of sugars (xylose, rhamnose, mannitol, and methyl α-D-mannopyranoside).

Table 1. Resources, the Number and Percentage of Samples Examined in This Study ^a

Sources	Number of Sample	Number of Isolated Bacteria
Dairy
Cheese	70 (4.65)	5 (1.7)
Cream	20 (7.18)	3 (15)
Curd	17 (9.15)	3 (6.17)
Total	107	11 (2.10)
Processing Meat
Sausage	90 (9.42)	6 (6.6)
Meat extracts	55 (2.26)	4 (2.7)
Chicken extract	65 (9.30)	3 (6.4)
Total	210	13 (1.6)

^a Values are presented as No. (%).

3.2. Antimicrobial Susceptibility Test

Antibiotic susceptibility test was done by the disk diffusion method (Kirby Bauer) (16). The turbidity of broth after incubation was adjusted with sterile saline to achieve turbidity comparable to 0.5 McFarland standards.

Results were interpreted according to the clinical and laboratory standards institute (guidelines M45-A2). Eight antibiotic disks, including penicillin G (10U), chloramphenicol (10 μg), tetracycline (25 μg), trimethoprim (5 μg), streptomycin (10 μg), ampicillin (10 μg), ciprofloxacin (5 μg), and erythromycin (15 μg) (Himedia, India) were used. L. monocytogenes ATCC 7644 was used as the reference strain.

3.3. Multiple-Locus Variable Number of Tandem Repeat Analysis Typing

DNA extraction was prepared from full-grown colonies at 37°C overnight in brain heart infusion broth (BHI) by using a DNA extraction kit protocol (Roche Co, New York, USA). Five reference loci (Lm10, Lm11, Lm23, Lm32, and LM-TR6) and 5 corresponding primer pairs previously described (Table 2), were used to perform typing of L. monocytogenes strains by MLVA technique (17). Specificity of all primers was verified using the basic local alignment search tool (BLAST). PCR technique was used for amplification of each locus. The reaction mixture consisted of 2 μL of extracting DNA, 2.5 μL of 10 × PCR buffer, 1.5 μL MgCl2 (50 mM), 0.5 μL dNTP (10 mM), 1.25 μL of each primer, 0.4 μL of Taq DNA polymerase (5 U/μL) and deionized water to a final volume of 25 μL. The reaction mixture was amplified in a thermocycler (Eppendorf, Germany) in the following PCR conditions: denaturation at 94°C for 5 minutes, 31 cycles with denaturation at 94°C for 30 s, annealing at 52°C for 20 s, extension at 72°C for 45 seconds, and final extension at 72°C for 5 minutes. The results of PCR products were further analyzed by electrophoresis in 3% agarose gel for 120 minutes in tris-acetate buffer, visualized by ethidium bromide staining, illuminated by UV-Transilluminator and accepted by a gel documentation apparatus (UVP Gel Seq Software, England). A 50 bp DNA ladder (fermentas) was used as a size reference.

Table 2. Characteristics of Selected TR Loci for MLVA Sub-Typing of L. monocytogenes

VNTR Locus	Primers	TR Sequence	Protein Description or Function
Lm10	F-CAGATATCGATACGATTGAC	-GAAGAACCAAAA-	ATP-dependent metalloprotease FtsH
Lm10	R-CAGTTAGTATTTCCAACGTC	-GAAGAACCAAAA-	ATP-dependent metalloprotease FtsH
Lm11	F-GAATAAAATGCTAGATGTGG	-TTGCTTGTTTTTG-	Cell wall surface anchor family protein
Lm11	R-CCGATTCAAAAATAGTAAAC	-TTGCTTGTTTTTG-	Cell wall surface anchor family protein
Lm23	F-TATTTACGGAAAAGACGTAG	-CATCGG-	Putative peptidoglycan bound protein (LPXTG motif)
Lm23	R-CGTAACTGTCCTACCATTAG	-CATCGG-	Putative peptidoglycan bound protein (LPXTG motif)
Lm32	F-AAAGCTTTGCCAGTGCAAGT	-AACACC-	Hypothetical protein
Lm32	R-TTGTGACTTGGCACTTCTGG	-AACACC-	Hypothetical protein
LM-TR6	F-AAA AGC AGC GCC ACT AAC G-	-CCAGACCCAACA-	Hypothetical protein
LM-TR6	R-TAA AAA TCC CAA TAA CAC TCC TGA-	-CCAGACCCAACA-	Hypothetical protein

3.4. Calculation of the Number of Tandem

The following formula was used to calculate the number of tandem repeats (Equation 1):

(1)

The flanking regions with known sizes were previously described (11, 15). In addition, to calculate the PCR product molecular weight from the images obtained on the gel, GeneTools from SynGene Version 3. 08 Software were used (Figure 1).

Figure 1. Photo of GeneTools Software (Version 3. 08) Environment to Calculate the Size of the PCR Product

3.5. Charting of Phylogenic Tree

After converting the number of tandem repeat of each locus to the corresponding sequences of isolates, evolutionary strain diagram was drawn by using of MEGA 6.06 software.

4. Results

4.1. Bacterial Isolation

Out of 317 samples, 24 L. monocytogenes isolates were collected from the dairy and meat products (Table 1).

4.2. Antimicrobial Susceptibility Testing of L. monocytogenes

The response of L. monocytogenes to the various antimicrobial agents is listed in Table 3. High resistance to penicillin was found amongst isolates. Resistance to penicillin G among strains isolated from dairy samples and processing meat were 54.54% and 46.15%, respectively. Antibiotic resistance was not found for tetracycline, trimethoprim, ciprofloxacin, ampicillin, and erythromycin.

Table 3. Susceptibility of L. monocytogenes Strains to 8 Antimicrobial Agents ^a,b

Antibiotic	Dairy Samples (Total = 11)			Processing Meat Samples (Total = 13)			Total Samples (24)
	R	I	S	R	I	S	R	I	S
Chloramphenicol	1 (9.09)	4 (36.36)	6 (54.54)	0 (0)	6 (46.15)	7 (53.84)	1 (4.16)	10 (41.66)	13 (54.16)
Penicillin G	6 (54.54)	4 (36.36)	1 (9.09)	6 (46.15)	4 (30.76)	3 (23.07)	12 (50)	8 (33.33)	4 (16.66)
Streptomycin	1 (9.09)	2 (18.18)	8 (72.72)	0 (0)	6 (46.15)	7 (53.84)	1 (4.16)	8 (33.33)	15 (62.5)
Tetracycline	0 (0)	1 (9.09)	10 (90.90)	0 (0)	3 (23.07)	10 (76.9)	0 (0)	4 (16.66)	20 (83.33)
Trimethoprim	0 (0)	0 (0)	11 (100)	0 (0)	0 (0)	13 (100)	0 (0)	0 (0)	24 (100)
Ciprofloxacin	0 (0)	1 (9.09)	10 (90.90)	0 (0)	3 (23.07)	10 (76.9)	0 (0)	4 (16.66)	20 (83.33)
Ampicillin	0 (0)	1 (9.09)	10 (90.90)	0 (0)	1 (7.69)	12 (92.3)	0 (0)	2 (8.33)	22 (91.66)
Erythromycin	0 (0)	2 (16.6)	9 (81. 81)	0 (0)	1 (7.69)	12 (92.3)	0 (0)	3 (12.5)	3 (87.5)

^a Abbreviations: I, intermediate resistance; R, resistant; and S, susceptible.

^b Values are presented as No. (%).

4.3. Multiple Locus VNTR Analysis Typing

After analysis of gel photo PCR product by Genetools software, the number of tandem repeats was calculated by using the described formula (Table 4) and the number of repeats per locus isolates was typed. To this effect, strains that have 80% or more than 80% similarity were placed at one type and other strains were placed on the different types. Finally, 13 different types were obtained as well as type 2 with 6 strains and type 3 with 4 strains, respectively, which were the most abundant types (Table 4).

Table 4. Types, Number, Sources, and the Number of Tandem Repeats per Lux of L. Monocytogenes Isolates

Strain	Source	LM10	LM11	LM23	LM32	LM-TR6	Type Number
1	Cheese	3	4	28	19	0	1
2	Cheese	3	4	28	24	0	1
3	Cheese	4	0	28	21	0	5
4	Cheese	4	3	28	24	3	6
5	Cheese	5	3	30	23	3	2
6	Cream	5	3	30	15	3	2
7	Cream	5	3	30	15	3	2
8	Cream	5	3	31	15	3	2
9	Curd	5	3	0	15	3	7
10	Curd	4	3	31	15	3	3
11	Curd	4	3	31	21	3	3
12	Sausage	0	4	38	16	0	8
13	Sausage	4	3	31	16	3	3
14	Sausage	4	3	31	16	1	3
15	Sausage	5	3	30	16	1	2
16	Sausage	4	4	31	19	0	4
17	Sausage	4	4	31	19	0	4
18	Meat extracts	5	3	30	15	2	2
19	Meat extracts	4	4	31	19	0	4
20	Meat extracts	6	3	30	13	2	9
21	Meat extracts	5	1	30	22	2	10
22	Chicken extract	6	1	30	21	2	11
23	Chicken extract	5	2	30	22	3	12
24	Chicken extract	5	2	0	22	3	13

4.4. Phylogenic Tree

Evolutionary relationships tree of strains are shown in Figure 2.

Figure 2. Dendrogram of the MLVA Results for the L. monocytogenes Isolates

5. Discussion

The actual prevalence of Listeria monocytogenes in Iran is unknown and little information about L. monocytogenes is available. In addition, this disease has not been reported in the Iranian health system. In this study, the prevalence of L. monocytogenes was 7.5%. In a study in 2009 by Morobe (18), the prevalence of L. monocytogenes in food samples was reported at 3.4%, which was not consistent with our study. In addition, in a study during 2002 - 2004 conducted in Brazil, the bacteria were isolated from 3.4% of food samples (19). Arslan and colleagues studied the prevalence of L. monocytogenes in Turkish white cheese house-cooked. The overall prevalence of various strains of Listeria in cheese was 1.33% and L. monocytogenes was isolated from 2.9% of the samples (20). In the study by Jalali et al. on the frozen meat, fresh meat, beef and mutton samples, totally 4 (2.1%) L. monocytogenes were isolated (21).

In Northern Ireland in 2003, 205 samples of poultry meat packed two large sales were made in 14 cases (18%) of L. monocytogenes was isolated (125) that corresponded with the present study. These various findings could be due to differences in sample size, geographic area, or processing of food production. Until now, it seemed that L. monocytogenes remains sensitive to the antibiotics used to treat listeriosis, however, in recent years, resistant strains of bacteria to antibiotics have been reported (22).

In the present study, L. monocytogenes strains isolated from food samples were susceptible to trimethoprim, erythromycin, ciprofloxacin, ampicillin, and tetracycline. The highest sensitivity (100%) was to trimethoprim. In addition, strains resistant to penicillin (50%) were observed. Prazak and Murano in their study reported the isolation of bacteria susceptible to all antibiotics except penicillin, which corresponds to the results of our study (23). Arslan and Ozdemir (20) in their study reported resistance to penicillin and chloramphenicol, which are in agreement with the results of the present study. Morvan et al. (24) did not report any case of resistance to penicillin in France (24). Penicillin resistance in our study (but susceptibility to these antibiotics in Morvan et al. (24) study) is possibly due to differences in drug use practices between two countries and the indiscriminate use of antibiotics, and sometimes without a doctor's prescription is in our country.

In the present study, we used the MLVA technique. In this study, 5 loci (Lm10, Lm11, Lm 23, Lm 32, and LM-TR6) were used for MLVA-typing. By using this technique, strains were successfully differentiated from each other and 13 different types were obtained. Type 2 with 6 strains and type 3 with 4 strains were the most abundant types, respectively. In this study, some strains isolated from cheese, cream, and sausage were at one type (type 1), also some strains isolated from meat and meat extracts were at one type (type 4). These findings show that the source of infection could be the same. In addition, strains isolated from chicken extracts were in different types, which show the different sources of pollution.

In 2006, Murphy and colleagues, for the first time were able to differentiate 45 foodborne L. monocytogenes strains using MLVA (11). They used 6 loci and their results were consistent with the results of current study. In another study, Chen et al. (14) and colleague isolated a total of 46 strains of L. monocytogenes from food samples and typed them by MLVA. Their results were in agreement with the results of the present study too. The results of our study show the high frequency of L. monocytogenes in dairy and meat products and resistance to penicillin is significant too. Processing of these foods to eliminate this bacterium is not suitable, and consumption of these foods for people at risk, especially pregnant women is very dangerous. Moreover, because penicillin with gentamicin is the treatment of choice for listeriosis in Iran and other countries, antibiotic susceptibility testing for on time and precise treatment is crucial. Given the importance of identifying potential sources of pollution, particularly the incidence of epidemics, utilizing a quick and inexpensive technique with high contrast resolution is necessary. In this regard, MLVA technique is perfect for these purposes, and is recommended due to its simplicity. According to study conducted by Noller et al. (25), MLVA method has a sensitivity equal to that of PFGE and a specificity superior to that of PFGE in outbreak detection.

MLVA analysis showed that samples obtained from different sources could have similar genetic profile. As a result, administration of penicillin in patients with listeriosis (especially pregnant women) is suggested and antibiotic susceptibility test recommended. The fast and accurate methods such as MLVA for tracking of pollution sources of L. monocytogenes are recommended during outbreaks because of its ideal properties, high sensitivity, and specificity comparing with other methods.

Acknowledgements

Footnotes

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