Phenotypic and genotypic characteristics of Listeria monocytogenes isolated from domestic animals

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Phenotypic and genotypic characteristics of Listeria monocytogenes isolated from domestic animals

1. Introduction

Listeria monocytogenes is an omnipresent bacterium that can cause severe aggressive disease in humans and animals [1].

2. Materials and methods

2.1. Bacterial strains

During November 2010 - December 2013 a total of 342 domestic animal samples were collected from Tehran University Veterinary Clinic, Iran (Table 1).

2.2. Microbiological and biochemical methods

5ml of broth specimen (included blood and urine) inoculated into TSBYE (Tryptic soy broth positive 0.6% yeast extract, Merck, Germany). after incubation all specimens at 4°C and Later 7-16 days still 6 months incubation, samples were cultured on PALKAM Agar (Merck, Germany) and Listeria selective agar (Himedia, India) and plates were incubated at 35°C for 24-48 h. Almost 5-6 suspend grown colonies from both culture media were injected into Brain Heart Infusion agar (Merck, Germany) and recognized using microbiological and biochemical tests such as gram’s 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 - D- mannopyranoside).

2.3. Antimicrobial susceptibility of L. monocytogenes

Antibiotic susceptibility was done by the disk diffusion method (Bauer, Kirby, Sherris, & Turk, 1966). The turbidity of broth after incubation was adjusted with sterile saline to achieve turbidity comparable of 0.5 McFarland standard. results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI, 2006).Ten antibiotic discs, namely penicillin G (10U),chloramphenicol (10 μg), tetracycline (25 μg), trimethoprim (5 μg), streptomycin (10 μg), ampicillin (10 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), cephotaxim (30 μg), erythromycin (15 μg) (Himedia, India) were used. L. monocytogenes ATCC 7644 was used as the reference strain.

2.4. DNA isolation

DNA extraction 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).

2.5. Multiple-locus variable number of tandem repeat analysis (MLVA) typing

Five reference loci (Lm-10, Lm-11, Lm-23, Lm-32, lm-TR6) and Five corresponding primer pairs that previously described [2] (Table 2), were used to perform typing of L. monocytogenes strains by MLVA Technique. Specificity of all primers was verified using the Basic Local Alignment Search Tool (BLAST). PCR technique was used for amplification of locus. The reaction mix 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 m M), 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 thermo cycler (Eppendorf, Germany) with the following PCR conditions: Denaturation at 94°C for 5 min, 31 cycles with denaturation at 94°C for 30 s, annealing at 52°C for 20 s, extension at 72°C for 45 s and final extension at 72°C for 5 min. The results of PCR products were further analyzed by electrophoresis in 3% agarose gel for 120 min in Tris-acetate buffer, visualized by ethidium bromide staining, illuminated by UV- Trans illuminator and accepted by a gel documentation apparatus (UVP Gel Seq Software, England). A 50 bp DNA ladder (fermentase) was used as a size reference.

2.6. Calculate the number of tandem repeats

The following formula was used to calculate the number of tandem repeats:

Number of

The flanking regions with known sizes that Previously described (3,4). Also, to calculate the PCR product molecular weight from the images obtained on the gel, GeneTools from SynGene Version 3. 08 Software were used (Fig.1).

2.7. Statistical analysis

Statistical analysis performed after collected data with use SPSS (14.0 -Version) and Excel 2013 and done with ki2 (Æ™ Square Pierson) Statistical exam

3. Result

3.1. Bacterial isolation

In this study a total of 24 isolates of L. monocytogenes from a total 342 specimen include Blood and urine samples Of domestic animals (goats, sheep and Cattle) from Tehran University Veterinary Clinic, Iran were obtained (table 1).

3.2. Antimicrobial susceptibility testing of L.monocytogenes

The response of L. monocytogenes to the various antimicrobial agents is presented in Table 3. High resistance to penicillin and Cephotaxim were found amongst isolates. Resistance to penicillin G Among isolated from goat, sheep and cattel was %50, %50 and %75, and to the Cephotaxim was %50, %75 and %50 respectively. Antibiotic resistance was not found for Trimethopirim and Ciprofloxacin In cattle isolates.

3.3. MLVA typing

After analysis of gel photo PCR product by Gene tools softwar , the number of by using the formula described above, was calculated (table4) and by helping the number of repeats per locus isolates were typed. For this purpose, strains that have 80 percent or more than 80 percent Similarity Was placed at one type and other strains placed on the different types. Finally, 13 different types obtained as well as type 2 with 6 strains and type 3 with 4 strains, respectively, were the most abundant types (table5).

After converting the number of tandem repeat of each locus to the corresponding sequences of isolates,

1-Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-borne pathogen. Microbiology and Molecular Biology Reviews 55, 476–511.

2- Identification of an optimized panel of variable number tandem-repeat (VNTR) loci

for Listeria monocytogenes typing

Xiujuan Li a,1, Bixing Huang b,⁎,1, Sofroni Eglezos c, Trudy Graham b, Barry Blair b, John Bates b

3-Development and application of Multiple-Locus Variable Number

of tandem repeat Analysis (MLVA) to subtype a collection

of Listeria monocytogenes

Mary Murphy a,b, Deborah Corcoran c, James F. Buckley a, Micheál O'Mahony d,

Paul Whyte b, Séamus Fanning b,⁎

4-Multiple-Locus Variable-Number Tandem-Repeat Analysis as a Tool

for Subtyping Listeria monocytogenes Strains_Katharine E. Volpe Sperry,1* Sophia Kathariou,2 Justin S. Edwards,1 and Leslie A. Wolf1North Carolina State Laboratory of Public Health, Raleigh, North Carolina,1 and North Carolina State University,Raleigh, North Carolina2

2.3. Statistical analysis

Statistical analysis performed after collected data with use SPSS (14.0 -Version) and Excel 2013 and done with ki2 (Æ™ Square Pierson) Statistical exam

Table 1

Number (%) of isolates

Number of samples

Type of livestock

(% 9.6) 12

125

Goat

(% 6.7) 8

119

Sheep

(% 4.08) 4

98

Cattle

Table 3. Susceptibility of L. monocytogenes to 10 antimicrobial agents.

Antibiotic

No. (%) from Goat

No. (%)from Sheep

No. (%) from Cattle

Total = 12

Total = 8

Total = 4

R

I

S

R

I

S

R

I

S

Chloramphenicol

2(16.66)

4(33.33)

6(50)

0(0)

4(50)

4(50)

1(25)

1(25)

2(50)

Penicillin G

6(50)

4(33.33)

2(16.66)

4(50)

2(25)

2(25)

3(75)

1(25)

0(0)

Streptomycin

2(16.66)

2(16.66)

8(66.66)

0(0)

4(50)

4(50)

0(0)

1(25)

3(75)

Tetracycline

0(0)

2(16.6)

10(83.33)

0(0)

2(25)

6(75)

0(0)

1(25)

3(75)

Trimethopirim

0(0)

0(0)

12(100)

0(0)

0(0)

8(100)

0(0)

0(0)

4(100)

Ciprofloxacin

0(0)

2(16.66)

10(83.33)

0(0)

2(25)

6(75)

0(0)

0(0)

4(100)

Ampicillin

0(0)

1(8.33)

11(91.66)

0(0)

1(12.5)

7(87.5)

0(0)

1(25)

3(75)

Cephotaxim

6(50)

0(0)

6(50)

6(75)

0(0)

2(25)

2(50)

1(25)

1(25)

Norfloxacin

2(16.66)

0(0)

10(83.33)

2(25)

2(25)

4(50)

1(25)

1(25)

2(50)

Erythromycin

0(0)

2(16.6)

10(83.33)

0(0)

1(12.5)

7(87.5)

0(0)

1(25)

3(75)

No.: number of isolates; R: resistant; I: intermediate resistance; S: susceptible.

Table 2

VNTR locus

Primers

TR Sequence

Protein description or function

Lm10

F-CAGATATCGATACGATTGAC

R-CAGTTAGTATTTCCAACGTC

-GAAGAACCAAAA-

ATP-dependent metalloprotease

FtsH

Lm11

F-GAATAAAATGCTAGATGTGG

R-CCGATTCAAAAATAGTAAAC

-TTGCTTGTTTTTG-

Cell wall surface anchor family

protein

Lm23

F-TATTTACGGAAAAGACGTAG

R-CGTAACTGTCCTACCATTAG

-CATCGG-

Putative peptidoglycan bound

protein (LPXTG motif)

Lm32

F-AAAGCTTTGCCAGTGCAAGT

R-TTGTGACTTGGCACTTCTGG

-AACACC-

Hypothetical protein

LM-TR6

F-AAA AGC AGC GCC ACT AAC G-

R-TAA AAA TCC CAA TAA CAC TCC TGA-

-CCAGACCCAACA-

Hypothetical

protein

Fig.1

Table 4

Strain

Source

LM10

LM11

LM23

LM32

LM-TR 6

1

Cattle

3

4

28

19

0

2

Cattle

3

4

28

24

0

3

Cattle

4

0

28

21

0

4

Cattle

4

3

28

24

3

5

Sheep

5

3

30

23

3

6

Sheep

5

3

30

15

3

7

Sheep

5

3

30

15

3

8

Sheep

5

3

31

15

3

9

Sheep

5

3

0

15

3

10

Sheep

4

3

31

15

3

11

Sheep

4

3

31

21

3

12

Sheep

0

4

38

16

0

13

Goat

4

3

31

16

3

14

Goat

4

3

31

16

1

15

Goat

5

3

30

16

1

16

Goat

4

4

31

19

0

17

Goat

4

4

31

19

0

18

Goat

5

3

30

15

2

19

Goat

4

4

31

19

0

20

Goat

6

3

30

13

2

21

Goat

5

1

30

22

2

22

Goat

6

1

30

21

2

23

Goat

5

2

30

22

3

24

Goat

5

2

0

22

3

table5

13

12

11

10

9

8

7

6

5

4

3

2

1

types

24

23

22

21

20

12

9

4

3

16,17,19

10,11,13,14

5,6,7,8,15,18

1,2

strain