Phenotypic and genotypic characteristics of Listeria monocytogenes isolated from domestic animals Essay

Phenotypic and genotypic features of Listeria monocytogenes isolated fromdomesticanimate beings

1. Introduction

Listeria monocytogenes is an ubiquitous bacteria that can do terrible aggressive disease in worlds and animate beings [ 1 ] .

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2. Materials and methods

2.1. Bacterial strains

During November 2010 – December 2013 a sum of 342 domestic animate being samples were collected from Tehran University Veterinary Clinic, Iran ( Table 1 ) .

2.2. Microbiological and biochemical methods

5ml of broth specimen ( included blood and piss ) inoculated into TSBYE ( Tryptic soy broth positive 0.6 % barm infusion, Merck, Germany ) . after incubation all specimens at 4°C and Subsequently 7-16 yearss still 6 months incubation, samples were cultured on PALKAM Agar ( Merck, Germany ) andListeriasselective agar ( Himedia, India ) and home bases were incubated at 35°C for 24-48 h. Almost 5-6 suspend grown settlements from both civilization media were injected into Brain Heart Infusion agar ( Merck, Germany ) and recognized utilizing microbiological and biochemical trials such as gram’s staining, catalase reaction, oxidase trial, haemolysis on Sheep Blood Agar, Christie Atkins Munch Petersen ( CAMP ) trial, Voges – Proskauer ( MR-VP ) , Methyl Red trials, and agitation of sugars ( xylose, rhamnose, Osmitrol and – methyl – D- mannopyranoside ) .

2.3. Antimicrobial susceptibleness of L. monocytogenes

Antibiotic susceptibleness was done by the disc diffusion method ( Bauer, Kirby, Sherris, & A ; Turk, 1966 ) . The turbidness of stock after incubation was adjusted with unfertile saline to accomplish turbidness comparable of 0.5 McFarland criterion. consequences were taken harmonizing to the Clinical and Laboratory Standards Institute ( CLSI, 2006 ) .Ten antibiotic phonograph record, viz. penicillin G ( 10U ) , Chloromycetin ( 10 ?g ) , Achromycin ( 25 ?g ) , trimethoprim ( 5 ?g ) , streptomycin ( 10 ?g ) , Principen ( 10 ?g ) , Cipro ( 5 ?g ) , norfloxacin ( 10 ?g ) , cephotaxim ( 30 ?g ) , erythromycin ( 15 ?g ) ( Himedia, India ) were used. L. monocytogenes ATCC 7644 was used as the mention strain.

2.4. Deoxyribonucleic acid isolation

Deoxyribonucleic acid extraction prepared from adult settlements at 37°C overnight in Brain Heart Infusion broth ( BHI ) by utilizing a Deoxyribonucleic acid extraction Kit protocol ( Roche Co, New York, USA ) .

2.5. Multiple-locus variable figure of tandem repetition analysis ( MLVA ) typing

Five mention venue ( Lm-10, Lm-11, Lm-23, Lm-32, lm-TR6 ) and Five matching primer braces that antecedently described [ 2 ] ( Table 2 ) , were used to execute typewriting ofL. monocytogenesstrains by MLVA Technique. Specificity of all primers was verified utilizing the Basic Local Alignment Search Tool ( BLAST ) . PCR technique was used for elaboration of venue. The reaction mix consisted of 2 µl of pull outing DNA, 2.5 µl of 10 ? PCR buffer, 1.5 µl MgCl2 ( 50 millimeter ) , 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 H2O to a concluding 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 rhythms with denaturation at 94°C for 30 s, tempering at 52°C for 20 s, extension at 72°C for 45 s and concluding extension at 72°C for 5 min. The consequences of PCR merchandises were farther analyzed by cataphoresis 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 certification setup ( UVP Gel Seq Software, England ) . A 50 bp DNA ladder ( fermentase ) was used as a size mention.

2.6.Calculate the figure oftandem repetitions

The undermentioned expression was used to cipher the figure of tandem repetitions:

Number of

The flanking parts with known sizes that Previously described ( 3,4 ) . Besides, to cipher the PCR merchandise 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 information with usage SPSS ( 14.0 -Version ) and Excel 2013 and done with qi2(?™Square Pierson ) Statistical test

3. Consequence

3.1. Bacterial isolation

In this survey a sum of 24 isolates of L. monocytogenes from a entire 342 specimen include Blood and urine samples Of domestic animate beings ( caprine animals, sheep and Cattle ) from Tehran University Veterinary Clinic, Iran were obtained ( table 1 ) .

3.2.Antimicrobial susceptibleness testing of L.monocytogenes

The response of L. monocytogenes to the assorted antimicrobic agents is presented in Table 3. High opposition to penicillin and Cephotaxim were found amongst isolates. Resistance to penicillin G Among isolated from caprine animal, sheep and cattel was % 50, % 50 and % 75, and to the Cephotaxim was % 50, % 75 and % 50 severally. Antibiotic opposition was non found for Trimethopirim and Ciprofloxacin In cattle isolates.

3.3. MLVA typing

After analysis of gel exposure PCR merchandise by Gene tools softwar, the figure ofby utilizing the expression described above, was calculated ( table4 ) and by assisting the figure of repetitions per venue isolates were typed. For this intent, strains that have 80 per centum or more than 80 percent Similarity Was placed at one type and other strains placed on the different types. Finally, 13 different types obtained every bit good as type 2 with 6 strains and type 3 with 4 strains, severally, were the most abundant types ( table5 ) .

After change overing the figure of tandem repetition of each venue 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 figure tandem-repeat ( VNTR ) venue

for Listeria monocytogenes typing

Xiujuan Li a,1, Bixing Huang B, a?Z,1, Sofroni Eglezos degree Celsius, Trudy Graham B, Barry Blair B, John Bates B

3-Development and application of Multiple-Locus Variable Number

of tandem repetition Analysis ( MLVA ) to subtype a aggregation

of Listeria monocytogenes

Mary Murphy a, B, Deborah Corcoran degree Celsius, James F. Buckley a, Micheal O’Mahony vitamin D,

Paul Whyte B, Seamus Fanning B, a?Z

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 information with usage SPSS ( 14.0 -Version ) and Excel 2013 and done with qi2(?™Square Pierson ) Statistical test

Table 1

Number ( % ) of isolates

Number of samples

Type of farm animal

( % 9.6 ) 12

125

Goat

( % 6.7 ) 8

119

Sheep

( % 4.08 ) 4

98

Cattles

Table 3.Susceptibility ofL. monocytogenesto 10 antimicrobic agents.

Antibiotic

No. ( % ) from Goat

No. ( % ) from Sheep

No. ( % ) fromCattles

Entire = 12

Entire = 8

Entire = 4

Roentgen

I

Second

Roentgen

I

Second

Roentgen

I

Second

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. : figure of isolates ; R: resistant ; I: intermediate opposition ; S: susceptible.

Table 2

VNTR venue

Primers

TR Sequence

Protein description or map

Lm10

F-CAGATATCGATACGATTGAC

R-CAGTTAGTATTTCCAACGTC

-GAAGAACCAAAA-

ATP-dependent metalloprotease

FtsH

Lm11

F-GAATAAAATGCTAGATGTGG

R-CCGATTCAAAAATAGTAAAC

-TTGCTTGTTTTTG-

Cell wall surface anchor household

protein

Lm23

F-TATTTACGGAAAAGACGTAG

R-CGTAACTGTCCTACCATTAG

-CATCGG-

Putative peptidoglycan edge

protein ( LPXTG motive )

Lm32

F-AAAGCTTTGCCAGTGCAAGT

R-TTGTGACTTGGCACTTCTGG

-AACACC-

Conjectural protein

LM-TR6

F-AAA AGC AGC GCC ACT AAC G-

R-TAA AAA TCC CAA TAA CAC TCC TGA-

-CCAGACCCAACA-

Conjectural

protein

Fig.1

Table 4

Strain

Beginning

LM10

LM11

LM23

LM32

LM-TR 6

1

Cattles

3

4

28

19

0

2

Cattles

3

4

28

24

0

3

Cattles

4

0

28

21

0

4

Cattles

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