Molecular Characterization of Multi-drug Resistant

Molecular Characterization of Multi-drug Resistant Enterococcus cecorum Isolated from Poultry in Bangladesh

M.Sc. Thesis A dissertation Submitted to the Department of Microbiology, Jagannath University, in Partial Fulfillment of the Requirement for the Degree of Master of Science in Microbiology

Submitted by-

Department of Microbiology

Roll No. M130605008

Faculty of Life and Earth Sciences

Registration No. M130605008

Jagannath University

Session: 2013-2014

Dhaka-1100, Bangladesh

December 2016

To My Beloved Parents

ACKNOWLEDGEMENT All praises are due to almighty Allah who enables me to complete this research work successfully in time. Without Allah‟s endless kindness and blessing it would not have been possible to finalize this capacious episode.

I extend my sincere appreciation deep and heartfelt gratitude to my respected teacher and supervisor Dr. Md. Zakaria Mia, Professor, Department of Microbiology, Jagannath University, for his valuable teacher‟s suggestion throughout the period of the research work and during the preparation of this research paper.

My profound gratitude to my supervisor Dr. Mohammed Abdus Samad, Senior Scientific Officer, Animal Health Research Division, Bangladesh Livestock Research Institute, for his continuous guidance, cooperation and scholastic inspiration.

It is a great pleasure to express my sincere appreciation to Dr. Shamima Begum, Professor and Chairmen, Department of Microbiology, Jagannath University, for her inspiration throughout this research work.

I am pleasure to express my cordial appreciation to all of my well wisher, friends and other relatives for their encouragement and aspiration during my study, research work and preparation of the project.

And finally my accolade to my parents who brought me to this stage, who dreamed me to perform best in life by manifesting eternal characters and prayed for my success every time. The Author December 2016

ABSTRACT

Molecular characterization, antimicrobial tolerance and multi-drug resistant pattern of isolated Enterococcus cecorum from poultry origin were investigated in current study. A total of 136 samples were collected from different poultry flock in Bangladesh including commercial broiler (n=36), commercial layer (n=69), and broiler breeder (n=31). Enterococcus cecorum represented 32 (24%) of all collected isolates and recorded as the first time isolation of this organism in Bangladesh. Prevalence of Enterococcus cecorum in commercial broiler (35%) was higher than the commercial layer (25%) and broiler breeder (13%). Increased level of antimicrobial resistance was observed to penicillin-G (95%), ceftriaxone (81%), erythromycin (57%), and amoxicillin (48%) in disk diffusion antimicrobial assay. Most isolates (18/21) showed multidrug resistance (MDR). Among eight tested antibiotics all isolates were resistant to azithromycin and sulfamethoxazole in minimum inhibitory concentration (MIC) test. Higher MIC50/ MIC90 (µg/ml) value were also observed to ceftriaxone (≥1024/ ≥1024), chloramphenicol (32/ 256), and gentamicin (8/ 128). In both kirby-bauer disk diffusion and MIC antimicrobial assay ciprofloxacin and gentamicin were more effective than the others. In phylogenetic analysis genetic variation among partially sequenced sodA gene has been observed. The presence of Enterococcus cecorum in Bangladeshi poultry and their MDR profile has been found as vulnerable. Further study on its pathogenesis, treatment and control is required immediately.

Keywords: Enterococcus cecorum, Kirby-Bauer disk diffusion, Multi-drug resistance, Minimum inhibitory concentration, sodA gene, Partial sequencing

I

TABLE OF CONTENTS ABSTRACT -------------------------------------------------------------------------------------------I TABLE OF CONTENTS ------------------------------------------------------------------------- II LIST OF FIGURES ------------------------------------------------------------------------------ VI LIST OF TABLES -------------------------------------------------------------------------------- IX ABBREVIATIONS ---------------------------------------------------------------------------------X SYMBOLS ---------------------------------------------------------------------------------------- XII CHAPTER 1 – INTRODUCTION -------------------------------------------------------------- 1 1.1 Overview ------------------------------------------------------------------------------------------ 1 1.2 Specific study objectives ----------------------------------------------------------------------- 3 CHAPTER 2 – REVIEW OF LITERATURES ---------------------------------------------- 4 2.1 Introduction --------------------------------------------------------------------------------------- 4 2.2 Properties and Characteristics of Enterococcus cecorum (EC) --------------------------- 5 2.2.1 Phenotypic Characteristics -------------------------------------------------------------- 5 2.2.2 Physical and Chemical growth properties --------------------------------------------- 5 2.2.3 Taxonomic Classification ---------------------------------------------------------------- 6 2.2.4 EC vs Other Enterococcus --------------------------------------------------------------- 6 2.3 Identification of Enterococcus cecorum (EC) ----------------------------------------------- 7 2.3.1 Presumptive Identification --------------------------------------------------------------- 7 2.3.1.1 Bacteriological Growth Medium------------------------------------------------- 7 2.3.1.2 Biochemical Tests------------------------------------------------------------------ 8 2.3.2 Molecular Methods ----------------------------------------------------------------------- 9 2.3.2.1 16s rDNA based Identification --------------------------------------------------- 9 2.3.2.2 sodA gene Specific Identification ----------------------------------------------- 9 2.3.2.3 Genus and Species specific Multiplex PCR ------------------------------------ 9 2.3.2.4 Pulsed Field Gel Electrophoresis and tDNA typing -------------------------- 9 2.4 Significance of Enterococcus cecorum (EC) ----------------------------------------------- 10 2.4.1 Virulence/ Pathogenecity --------------------------------------------------------------- 10 2.4.1.1 Animal Infection ------------------------------------------------------------------ 10 2.4.1.2 Arthritis in Poultry ---------------------------------------------------------------- 11

Contents

2.4.1.3 Spondylitis infection -------------------------------------------------------------- 12 2.4.1.4 Enterococcal Vertibral Osteoarthritis (EVOA) ------------------------------- 12 2.4.1.5 Femoral Head Necrosis (FHN) -------------------------------------------------- 14 2.4.1.6 EC Osteomyelitis ------------------------------------------------------------------ 15 2.4.1.7 Infection Dose --------------------------------------------------------------------- 15 2.4.2 Enterococcus cecorum (EC) Outbreak------------------------------------------------ 16 2.4.3 Transmission and Spread of EC ------------------------------------------------------- 16 2.4.4 Symptoms --------------------------------------------------------------------------------- 17 2.4.5 Treatment of EC infection -------------------------------------------------------------- 18 2.4.6 Human Infection ------------------------------------------------------------------------- 18 2.4.7 Antimicrobial resistance ---------------------------------------------------------------- 19 CHAPTER 3 - MATERIALS AND METHODS -------------------------------------------- 20 3.1 Outline of the Research study ----------------------------------------------------------------- 20 3.2 General Procedures and materials ------------------------------------------------------------ 21 3.2.1 Sterilization ------------------------------------------------------------------------------- 21 3.2.2 Preparation of solutions ----------------------------------------------------------------- 21 3.2.3 Growth of microorganisms ------------------------------------------------------------- 21 3.2.4 Maintenance of Media, Reagents and Solutions ------------------------------------ 21 3.2.5 Culture Media ---------------------------------------------------------------------------- 22 3.2.5.1 Complex media -------------------------------------------------------------------- 22 3.2.5.2 Selective media -------------------------------------------------------------------- 22 3.3 Origin of Samples ------------------------------------------------------------------------------ 23 3.4 Collection and Processing of Samples ------------------------------------------------------- 24 3.5 Selective Culture -------------------------------------------------------------------------------- 25 3.5.1 Culture in Kanamycin aesculin azide (KAA) agar ---------------------------------- 25 3.5.2 Culture in Blood agar-------------------------------------------------------------------- 26 3.5.3 Pure culture preservation --------------------------------------------------------------- 27 3.6 Biochemical tests ------------------------------------------------------------------------------- 28 3.6.1 Gram-staining ---------------------------------------------------------------------------- 28 3.6.2 Catalase test ------------------------------------------------------------------------------- 28 3.6.3 Esculin hydrolysis ----------------------------------------------------------------------- 28 3.6.4 Pyrrolidonyl Arylamidase (PYR) Test ------------------------------------------------ 28 3.6.5 Sugar fermentation----------------------------------------------------------------------- 29 III

Contents

3.7 Antibiotic sensitivity assay -------------------------------------------------------------------- 30 3.7.1 Kirby-Bauer disk diffusion method --------------------------------------------------- 30 3.7.1.1 Preparation of turbidity standard ------------------------------------------------ 30 3.7.1.2 Culture inoculation---------------------------------------------------------------- 30 3.7.1.3 Application of antibiotic discs to inoculated agar plates -------------------- 31 3.7.1.4 Reading plates and interpreting results ---------------------------------------- 32 3.7.2 Minimal Inhibitory Concentration (MIC) assay: microdilution technique ------ 33 3.7.2.1 Preparation of antibiotic Stock solution --------------------------------------- 33 3.7.2.2 Preparation of antibiotic dilution range ---------------------------------------- 33 3.7.2.3 Preparation of inoculum ---------------------------------------------------------- 34 3.7.2.4 Incubation -------------------------------------------------------------------------- 35 3.7.2.5 Measurement of turbidity -------------------------------------------------------- 35 3.8 Molecular Identification ----------------------------------------------------------------------- 36 3.8.1 Conventional Polymerase Chain Reaction (PCR) technique ---------------------- 36 3.8.1.1 DNA extraction -------------------------------------------------------------------- 36 3.8.1.2 PCR mastermix preparation ----------------------------------------------------- 37 3.8.1.3 DNA amplification---------------------------------------------------------------- 38 3.8.1.4 Preparation of 1.8% Agarose gel ----------------------------------------------- 38 3.8.1.5 Gel electrophoresis---------------------------------------------------------------- 39 3.8.2 Partial sequencing of sodA gene. ------------------------------------------------------ 40 3.8.2.1 PCR amplification for partial sequencing. ------------------------------------ 40 3.8.2.2 Purification of amplified DNA -------------------------------------------------- 40 3.8.2.3 Quantification of DNA purity and concentration ----------------------------- 41 3.8.2.4 Sequencing of target DNA using Sanger sequencing method -------------- 42 3.8.2.5 Purification of Extended PCR products ---------------------------------------- 44 3.8.2.6 Sequence analysis: Capillary electrophoresis --------------------------------- 45 3.8.2.7 Processing of obtained sequence and interpretation. ------------------------- 46 3.8.3 Phylogenetic analysis of obtained sequences ---------------------------------------- 48 CHAPTER 4 – RESULTS ----------------------------------------------------------------------- 49 4.1 Bacterial isolates and identification results in Selective media -------------------------- 49 4.2 Biochemical test results ------------------------------------------------------------------------ 51 4.2.1 Gram staining ----------------------------------------------------------------------------- 51 4.2.2 Catalase test ------------------------------------------------------------------------------- 52 IV

Contents

4.2.3 Esculin hydrolysis test result ----------------------------------------------------------- 52 4.2.4 PYR Test ---------------------------------------------------------------------------------- 54 4.2.5 Sugar fermentation profile of Enterococcus cecorum (EC) isolates. ------------- 55 4.3 Antibiotic sensitivity assay result ------------------------------------------------------------ 56 4.3.1 Antibiotic resistance pattern in Kirby-Bauer disk diffusion ----------------------- 56 4.3.2 Antimicrobial susceptibility pattern of EC isolates in MIC test ------------------ 60 4.4 Conventional Polymerase Chain Reaction (PCR) result ---------------------------------- 63 4.5 Result of Partial sequencing of sodA gene -------------------------------------------------- 64 4.6 Phylogenetic analysis -------------------------------------------------------------------------- 66 CHAPTER 5 - DISCUSSION ------------------------------------------------------------------- 68 CHAPTER 6 – CONCLUSION----------------------------------------------------------------- 70 REFERENCE--------------------------------------------------------------------------------------- 71 APPENDICES -------------------------------------------------------------------------------------- 89

V

LIST OF FIGURES Figure no.

Title of the figure

Page

Figure 2.1

Broiler chicken suffering from Enterococcal arthritis

12

Figure 2.2

Vertibral abscess on the spinal cord resulting in EVOA

13

Figure 2.3

Stages of poultry Femoral head necrosis (FHN)

14

Figure 3.1

Study design and work flow

20

Figure 3.2

Sampling area

23

Figure 3.3

Samples collection from different source and area

24

Figure 3.4

KAA agar preparation and incubation in CO2 Incubator under 5% CO2 environment

25

Figure 3.5

Preparation of 5% sheep blood agar media; Sheep blood was collected aseptically from healthy sheep

26

Figure 3.6

Comparison of culture suspension with 0.5 McFarland standard solutions

31

Figure 3.7

Serial dilution of antibiotic solution in a Microtiter plate using 8-channel pipette

34

Figure 3.8

MIC determinations based on the plate reader reading (PowerWave-XS, BioTek, VT, US)

35

Figure 3.9

Gel Electrophoresis performing in Mupid®-One Electrophoresis System

39

Figure 3.10

NanoDrop 2000c software, DNA concentration and purity measurement

41

Figure 3.11

Stratagene Mx3005p (Stratagene California, CA, US) thermal cycler used for Cycle sequencing of sodA amplified DNA

42

Figures

Figure no.

Title of the figure

Figure 3.12

Installation of sample and other required reagents into DNA sequence analyzer and starting the sequence analysis

45

Figure 3.13

Chromatogram view of genetic analyzer data collection software 3.0 output file (.ab1) in Chromas 2.6.2 software

47

Figure 3.14

Sequence alignment and assembly in BioEdit Sequence Alignment editor 7.2.5 software

47

Figure 4.1

Growth characteristic of Isolated EC in Kanamycin aesculin azide (KAA) agar media

49

Figure 4.2

Growth characteristic (α-hemolysis with slightly green pigmentation) of isolated EC in Blood agar (BA) media

50

Figure 4.3

Microscopic view (gram positive cocci) of EC isolates after gram staining

51

Figure 4.4

Catalase test reaction (no bubble production) of EC isolates

52

Figure 4.5

Esculin hydrolysis (Blackening of Bile esculin agar slant) by EC isolates

52

Figure 4.6

PYR positive (non EC) and negative (EC) reaction showed in PYR agar growth after addition of PYR reagent

54

Figure 4.7

Four types of fermentation test were performed for suspected isolates

55

Figure 4.8

Zone of inhibition (ZI) of EC growth on Mueller-Hinton agar due to the antimicrobial effect of diffused antibiotics impregnated in each disk

56

Figure 4.9

Antibiotic resistance profile of three type isolates

58

Figure 4.10

Sensitivity profiles of each antibiotic against all isolates

59

VII

Page

Figures

Figure no.

Title of the figure

Page

Figure 4.11

Reading view of incubated microtiter plate used for (Minimal Inhibitory Concentration) MIC assay in (Gen5 Microplate Reader and Imager Software V2.0)

60

Figure 4.12

PCR positive isolates were observed as amplified DNA band near 480bp after the Gel electrophoresis of PCR product

63

Figure 4.13

Phylogenetic tree of sodA gene sequence of present study isolates and Genbank sequence

67

VIII

LIST OF TABLES Table no.

Title of the Table

Page

Table 2.1

Biochemical profile of EC culture

8

Table 3.1

Antibiotic discs used in this study

31

Table 3.2

Primer sets used for PCR reaction

37

Table 3.3

Master mixture preparation for conventional PCR

37

Table 3.4

Thermal cycling profile used for PCR reaction

38

Table 3.5

Cycle sequencing formula

43

Table 3.6

Thermal cycle profile used for cycle sequencing

43

Table 3.7

Description of sodA partial sequences of different organism with their sources

48

Table 4.1

Growth result of samples in Selective media

50

Table 4.2

Sequential biochemical test results of all selected isolates (Positive in selective media)

53

Table 4.3

Sensitivity of EC isolates against all six antibiotics

57

Table 4.4-A

Distribution of minimal inhibitory concentration (MICs: µg/ml) obtained by broth-dilution for 21 EC isolates (10CB, 7-CL and 4-BB) from three types of sources

61

Table 4.4-B

Overall distribution of minimal inhibitory concentration (MICs: µg/ml) obtained by broth-dilution for 21 EC against Eight antibiotics

62

Table 4.5

5 Purity and concentration of primarily amplified DNA samples before and after DNA purification

64

Table 4.6

Partial sequence of studied EC isolates

65

ABBREVIATIONS Abbreviations

Stands For

BLRI

Bangladesh Livestock Research Institute

BA

Blood Agar

BB

Broiler Breeder

BHI

Brain Heart Infusion

BLAST

Basic Local Alignment Search Tool

bp

Base Pair

CB

Commercial Broiler

CFU

Colony Forming Unit

CL

Commercial Layer

CLED

Cystine Lactose Electrolyte Deficient

CLSI

Clinical And Laboratory Standards Institute

DNA

Deoxyribonucleic Acid

dNTPs

Deoxyribonucleoside Triphosphate

e. g.

Examplia Gratia

EC

Enterococcus cecorum

ES

Enterococcal Spondylitis

EUCAST

European Committee On Antimicrobial Susceptibility Testing

EVOA

Enterococcal Vertibral Osteoarthritis

FHN

Femoral Head Necrosis

FHS

Femoral Head Separation

FHT

Femoral Head Transitional Degeneration

FTV

Free Thoracic Vertebra

g/gm.

Gram

h

Hour (S)

Abbreviations

Abbreviations

Stands For

i.e

that is

KAA

Kanamycin Aesculin Azide Agar

kb

Kilo Base

LB

Luria-Bertani

MDR

Multi-drug Resistance

MHA

Mueller-Hinton Agar

MIC

Minimum Inhibitory Concentration

min

Minute (s)

ml

Milliliter

ng

Nano gram (109)

NCBI

National Center For Biotechnology Information

PBS

Phosphate Buffered Saline

PCR

Polymerase Chain Reaction

PFGE

Pulsed Field Gel Electrophoresis

pᴴ

Negative Logarithm of Hydrogen Ion Concentration

psi

Pounds Per Square Inch

PYR

Pyrrolidonyl Aminopeptidase

rDNA

Ribosomal DNA

rpm

Revolution Per Minute

Sec/s

Second (s)

TBE

Tris/Borate/EDTA

TSA

Tryptic Soy Agar

TSB

Tryptic Soy Broth

UV

Ultra Violate Ray

vs

Versus

w/v

Weight/Volume XI

SYMBOLS Symbols

Stands For

&

And

˚C

Degree centigrade

>

Greater than

35 days old (Herdt et al., 2009; Stalker et al., 2010; Jung and Rautenschlein, 2014).

It was not until 2002 that EC was associated with clinical disease in poultry, when it was isolated from osteomyelitis lesions following outbreaks of lameness in broilers in Scotland (Wood et al., 2002) and in the Netherlands (Devriese et al., 2002). More recently, a succession of similar outbreaks of E. cecorum arthritis and osteomyelitis have occurred in broilers and broiler breeders in the United States (Aziz and Barnes, 2007), Belgium (Herdt et al., 2009), Canada (Stalker et al., 2010) and Hungary (Makrai et al., 2011).

2.4.1.7 Infection Dose Data on EC infection dose for chicken are not still specifically abundant. 3 month old chicken inoculated with 5 x 108 CFU of cecal isolate did not cause illness (Devriese et al. 15

Review of Literature

1983). But infection was established successfully in pecking ducks in Germany. Intravenous infection of 12 days old bird with 1.5 × 109 CFU showed 100% mortality within 2 days. Whereas orally infected bird with 1.5 × 108 and 8.5 × 105 CFU exhibited 67% and 6.7% mortality respectively (Jung et al., 2013).

2.4.2 Enterococcus cecorum (EC) Outbreak In 2002, EC-associated disease outbreaks in broiler flocks were reported for the first time (Devriese et al., 2002; Wood et al., 2002; Herdt et al., 2009; Kense and Landman, 2011; Robbins et al., 2012; Szeleszczuk et al., 2013). Further reports from broiler and broiler breeder flocks indicate the growing importance of EC infections for the poultry industry. In Germany, EC is considered to be one of the most important bacterial pathogens in broilers (personal communication with other poultry veterinarians). EC associated disease outbreaks were also reported from ducks in Germany (Metzner et al., 2010; Jung et al., 2012)Infections in broilers and broiler breeders by EC causing clinical disease, have increasingly been described in various countries in the Northern Hemisphere over the past decade (Aitchison et al., 2014).

Recurrent outbreaks of lameness in affected houses with subsequent flocks have exacerbated the impact of this disease (Gingerich, 2009; Herdt et al., 2009; Gregersen et al., 2010; Jansson et al., 2012). Several cases of EC-related arthritis and osteomyelitis were diagnosed in broilers and broiler breeders in the state of Georgia.11.0 Recently, EC has been recognized as an emerging avian pathogen, associated with spondylitis, femoral head necrosis, and osteomyelitis in broiler and broiler breeder flocks in Scotland (Wood et al., 2002), Holland, (Devriese et al., 2002) Belgium, (Herdt et al., 2009) and the United States (Aziz T, Barnes HJ: 2007, Is spondylitis an emerging disease in broiler breeders? World Poultry 23:44–45).

2.4.3 Transmission and Spread of EC Recently, poultry or domestic animals (cats, dogs) are thought to be a possible source for transmission leading to EC associated septicemia in humans (Greub et al., 1997; Warnke et al., 2015). The bacteria can be transmitted through the air/inhalation, the beak, skin wounds, sole lesions, or through the yolk. However, the most likely route is through the beak. It is stated that the bacteria from the intestinal tract can enter the bloodstream 16


during the first days of life of the chick. In this period, the intestinal wall is still somewhat permeable to bacteria. In the body, the Enterococcus bacteria spread through the bloodstream. The bacteria can adhere to the wall of the blood vessels. This happens in the small blood vessels of, for example, cardiac valves and joints (Erum, 2014; Bouwhuis, 2015).

2.4.4 Symptoms The first symptoms of the course of disease between 10-14 days of age are: 1. Suddenly lame, usually unilateral 2. From some animals to a few percent of a litter 3. Mostly roosters and the heaviest animals The reason that the best-growing animals, largely roosters, are affected by EC is because these animals have a higher gastro-intestinal tract charge (Erum, 2014). The symptoms of the course of disease between 14-40 days of age are; 1. Number of unilaterally lame animals increases 2. Increasing paralysis: „kinky backs‟ 3. Chickens are sitting on their hind feet facing forward 4. Are only able to move backwards 5. Next phase is lateral recovery position 6. Chickens will die due dehydration and exhaustion (Erum, 2014) Symptoms of injury autopsy; 1. Purulent arthritis, mainly form the knee and sometimes hock joints. 2. Purulent inflammation of bone and bone marrow (osteomyelitis) 3. Neck and head of the femur 4. Top of the shank, at the knee 5. In „kinky back‟, at the T4-T7 vertebra 6. Sometimes an inflammation of the pericardium The damage EC causes is a morbidity of 20%, mortality up to 10% which is mainly by selection, and a substantially increased rejection percentage in slaughter (Erum, 2014).

17


2.4.5 Treatment of EC infection Antibiotics: curative 

Most penicillins (Phenoxypen / amoxycillin); +/- 100% sensitive

Antibiotics: Preventive 

Lincomycin-Spectinomycin during the first 3 to 4 days of life



Very efficient; reduction of problems> 90%



Mechanism of action is not clear.



Oral medication of 20 mg amoxicillin per kg bodyweight

(Jung and Rautenschlein, 2014) (Bouwhuis, 2015)

2.4.6 Human Infection Many species from Enterococcus have been found to be implicated in various clinical conditions in Human, including septicemia, mastitis, enteritis, respiratory disease, and urinary tract infections (Quinn et al., 1994; Songer and Post, 2004). During investigations on the identification of intestinal streptococci and enterococci from animal hosts, It was found that EC was not limited to poultry (De Baere et al., 2000). EC belongs to opportunistic pathogens and may also play a role as causative agent of diseases in humans (nosocomial infections) (Greub et al., 1997; Warnke et al., 2015).

In 2000 EC was isolated as the etiologic agent of a continuous ambulatory peritoneal dialysis peritonitis episode in an alcoholic patient (De Baere et al., 2000). The agent is sporadically involved in peritonitis, septicemia and empyema in humans suffering from other underlying diseases or weakening factors such as alcohol abuse, liver cirrhosis, malnourishment and continuous ambulatory peritoneal dialysis. EC associated disease in humans were reported several times (Greub et al., 1997; De Baere et al., 2000; Hsueh et al., 2000; Woo et al., 2004).

Reports on poultry infections caused by E. cecorum are frequent, but reports on human infections are extremely rare. Medline database search for E. cecorum revealed only five reports on human infections, i.e., a case of thorax empyema (Woo et al., 2004), septicaemia (Greub et al., 1997), peritonitis (De Baere et al., 2000; Hsueh et al., 2000), and aortic valve endocarditis (Ahmed et al., 2011). Recently reports of EC infection in 18


human are increasing. In 2015 EC associated incisional hernia plate infection and the first case of urinary tract colonization due to EC was reported in Marseille, France (Delaunay et al., 2015). Resent reports of nosocomial infection due to Enterococcus cecorum isolated from a blood culture of a 75-year-old septic male patient was documented in Germany (Warnke et al., 2015).

2.4.7 Antimicrobial resistance Antimicrobial resistance is notorious in enterococci, and resistance genes to antimicrobial agents commonly used in chickens have been found in EC (Cauwerts et al., 2007). It is possible that the use of antimicrobials may be selecting for EC in general or for strains which have acquired resistance determinants to frequently used antimicrobials.

Recent report showed that EC isolates from infected chicken are frequently resistant to erythromycin, high-level streptomycin, tetracycline, and bacitracin in Ontario, Canada. They showed higher MIC value in gentamicin and enrofloxacin than control isolates. It was inferred that strains of the clone associated with clinical infection have a decreased susceptibility to those antimicrobials than commensal intestinal strains (Boerlin et al., 2012).

19

CHAPTER 3 - MATERIALS AND METHODS

3.1 Outline of the Research study The working strategy for selection, isolation, confirmation, antibiogram and molecular characterization of Enterococcus cecorum (EC) was as follows (Figure 3.1): Figure 3.1 Study design and work flow.

Sample collection

Sample processing & Storage

Antibiogram

Biochemical confirmation

DNA extraction

PCR amplification & confirmation

Sequence

Sequence

alignment

reading

Comparison of sequences with database

Selective culture

Pure culture isolation and Storage

Purification of amplified DNA

Partial sequencing of targeted DNA

Materials and Methods

3.2 General Procedures and materials 3.2.1 Sterilization All equipment and glassware were sterilized by autoclaving at 15psi for 20 min in STURDY autoclave (SA-300V, New Taipei City, Taiwan). Sterilized equipments were stored in oven drier (Binder ed-115, Tuttlingen, Germany). All growth media and special solutions were sterilized by autoclaving at 15psi for 15 min in the autoclave. Fresh agar plates were prepared at room temperature, stored at 4°C until use, and then dried at room temperature under aseptic conditions before inoculation of the microorganisms.

3.2.2 Preparation of solutions Accurate weights of the components of various solutions were measured using a Metter balance (METLER-HR-200, Switzerland) and for approximate weights a standard laboratory top pan balance was used. A pH meter was used for determining pH values of different solutions, media, etc.

3.2.3 Growth of microorganisms All cultures were inoculated and incubated (Binder CB-150 CO2 Incubator, Chelmsford, UK) under aseptic conditions. Due to microaerophilic nature of target organism, all tests and culture were performed under 5% CO2 environment.

3.2.4 Maintenance of Media, Reagents and Solutions Preservation and maintenance of different materials including stains, heat labile chemicals and reagents, etc. were carried out in a Samsung refrigerator model: 360LTR (Seoul, South Korea) at 4°C. Nutrient Agar (NA) slants were used for the routine maintenance of the pure bacterial strains .The pure bacterial cultures were stored at 4°C until use.

21


3.2.5 Culture Media Various differential, complex and selective media obtained commercially were used for the growth and characterization of the selected bacteria. The media were prepared in the laboratory as per direction of the manufacturers.

3.2.5.1 Complex media Various complex media such as nutrient agar (NA), Nutrient broth (NB), buffered peptone water, Brain heart infusion broth (BHI) etc. were used for the growth of Enterococci. Mueller-Hinton agar (MHA) and Luria-Bertani broth (LB) were used for antibiotic sensitivity test purpose.

3.2.5.2 Selective media Selective media are media based on either of the two categories above supplemented with growth-promoting or growth-inhibiting additives. Blood agar (BA) supplemented with antibiotics and Kanamycin aesculin azide agar (KAA) were used for the isolation and selection of Enterococcus cecorum (EC). Selective broth such as BHI supplemented with antibiotic was used for initial selective enrichment of Enterococci.

22


3.3 Origin of Samples Sample collection was performed from two broiler breeder and layer farms at Gazipur and different poultry slaughter houses in Dhaka city (Figure 3.2). Three types sample were collected such as cloacal sample from Broiler Breeder flock (BB), cloacal sample from Commercial Layer flock (CL), cecal sample from Commercial Broiler flock (CB) after slaughtering.

Figure 3.2 Sampling area

23


3.4 Collection and Processing of Samples Preparation of Phosphate buffer (PBS) stock solution: PBS was prepared by dissolving 8.0g NaCl, 0.2g of KCl, 1.44g Na2HPO4 and 0.24g of KH2PO4 in 800ml of distilled H2O. Solution pH was adjusted to 7.4 with HCl and the volume brought to 1L with distilled H2O. Cloacal sample collection: Approximately 5-10g of faecal content were collected using a sterile, cotton tipped applicator and impregnated in 1:1 PBS solution previously dispended into a sterile 50ml Falcon tube and stored on ice until they could be processed.

Cecal sample collection: Poultry cecum was identified by examining intestine (Figure 3.3) and cecal incision was mediated using sterile surgical blade. Approximately 5-10g cecal content was collected as the same way as cloacal sample and mixed with the PBS solution. Cecal content was collected within 20 min of slaughtering the chicken. Sample was stored at low temperature until further processing.

Figure 3.3 Samples collection from different source and area

24


Long-term storage of sample: Collected samples were grown in Brain heart infusion (BHI) broth (Biotec, Dorset, UK) for 24h at 37oC under 5% CO2 and formulated as 25% glycerol stock, vortexed and then stored at -80oC in a 1.5ml vials.

3.5 Selective Culture 3.5.1 Culture in Kanamycin aesculin azide (KAA) agar Culture Pre-enriched in BHI broth (Biotec, Dorset, UK) was streaked on KAA agar (Oxoid Ltd, Hampshire UK) and incubated at 37oC for 24h under 5% CO2 environment using CO2 incubator (Binder CB-150 CO2 Incubator, Chelmsford, UK) (Figure 3.4). Colonies observed as slightly gray, small and partially blackening the media were presumed as EC and subcultered in Nutrient agar (NA) media (Oxoid Ltd, Hampshire UK) for pure culture isolation.

Figure 3.4 KAA agar preparation and incubation in CO2 Incubator under 5% CO2 environment

Fresh KAA agar plates before inoculation

Binder CB-150 CO2 Incubator

25


3.5.2 Culture in Blood agar Pure culture isolates obtained previously from KAA was streaked on Blood agar medium (Oxoid Ltd, Hampshire UK) supplemented with 5% sheep blood, 10µg/ml colistin sulfate and 5µg/ml nalidixic acid. Cultured plates were then incubated at 37oC for 24h under 5% CO2 environment using CO2 incubator (Binder CB-150 CO2 Incubator, Chelmsford, UK). Characteristic colonies with α-hemolysis were selected and subcultured in Blood agar to isolate pure culture (Stalker et al., 2010).

Figure 3.5 Preparation of 5% sheep blood agar media; Sheep blood was collected aseptically from healthy sheep (Goat shed, BLRI, Savar, Dhaka 1341).

Trimming and shaving of jugular area of sheep

Swabbing of punction site with 70% ethanol

Blood collection from jugular vein of the sheep

Fresh Blood agar (5% sheep blood) plates

26


3.5.3 Pure culture preservation Isolated bacteria were cultured in (Brain heart infusion) BHI broth (Biotec, Dorset, UK) at 37oC under 5% CO2 environment. After 24h incubation period of growth, culture was stored at -80oC after preparing 25% glycerol stock in a 1.5ml vials for long-term preservation and -20oC for short-term preservation (Gherna, 1994).

27


3.6 Biochemical tests 3.6.1 Gram-staining A Gram stain was performed for each suspect isolate and viewed under digitally equipped light microscope (Olympus CX41, Olympus Corp., Tokyo, Japan) using oil immersion. Microscopic view of gram staining were imaged using microscope digital camera (Olympus DP20, Olympus Corp., Tokyo, Japan) attached with the microscope. Any Gram-negative and non-coccoid bacteria were eliminated from the study (Claus, 1992).

3.6.2 Catalase test For each isolate, one drop of 3% hydrogen peroxide (H2O2) was added to a slide smeared with a single colony. Catalase activity was scored as positive if H2O2 was degraded and negative if H2O2 was not degraded. Bubbling after the addition of H2O2 to the smear indicated degradation. Any isolates negative for catalase activity were presumed as non EC culture (Facklam, 1995).

3.6.3 Esculin hydrolysis Bile-esculin agar (HiMedia, Mumbai, India) medium was prepared as agar slants. Bile esculin medium contains esculin and peptone for nutrition and bile to inhibit Grampositive bacteria other than enterococci. Ferric citrate is added as a color indicator. An inoculum from a pure culture was transferred aseptically to a sterile tube of bile esculin agar and streaked along the slant but not stabbed. The inoculated tube is incubated at 37oC for 24h under 5% CO2. Abundant growth on the slant indicates a positive test for growth in the presence of bile. If growth is present, esculin hydrolysis can be observed if the medium has taken on an intense, chocolate brown coloration. Isolates with positive Esculin hydrolysis were selected as EC bacteria (Devriese et al., 1983)

3.6.4 Pyrrolidonyl Arylamidase (PYR) Test Pure bacterial inoculum from previous BHI (Biotec, Dorset, UK) culture was streaked on PYR agar (HiMedia, Mumbai, India) media plate and incubated at 37oC for 24h. One drop of PYR reagent (Himedia, Mumbai, India) was added directly to suspected surface growth on plate. Observe for colour change after 2 minutes. The PYR reagent reacts with 28


b-naphthylamine to form a red coloured schiffs base indicating a positive reaction. Negative PYR reaction was presumed as EC (Facklam et al., 1982; Devriese et al., 1983).

3.6.5 Sugar fermentation Phenol red stock solution preparation: Phenol red stock solution was prepared as .8mg/ml in distilled water.

Preparation of fermentation broth: Fermentation broth was prepared by adding 10ml of stock phenol red solution to prepare 100ml Buffered peptone water broth (HiMedia, Mumbai, India). 1gm desired sugar was added with the peptone water to prepare final fermentation broth. Prepared media was sterilized at 115o C for 15 minutes. After sterilization media was dispensed into sterile fermentation tubes and inoculated with pure bacterial culture. Inoculated media was incubated at 37oC for 18-24h. Color change of inoculated broth from red to yellow represented the fermentation of that sugar added in that broth.

Ribose, Raffinose, Lactose and Glycerol fermentation were assayed for each suspected culture. Isolates those had demonstrated positive Esculin hydrolysis, Ribose, Raffinose & Lactose fermentation and Negative Catalase & Glycerol fermentation were confirmed as positive EC culture and subjected for antimicrobial assay and molecular characterization.

29


3.7 Antibiotic sensitivity assay

3.7.1 Kirby-Bauer disk diffusion method 3.7.1.1 Preparation of turbidity standard McFarland 0.5 turbidity standards were prepared as per the standard guidelines described by the (Cockerill et al., 2010). A volume of 0.5 ml of a 1.175% (w/v) barium chloride dihydrate (BaCl2.2H2O) solution was added to 99.5 ml of 0.18mol/L (1% v/v) sulfuric acid with constant stirring to maintain the suspension. The turbidity standard was then aliquoted into test tubes, identical to those used to prepare the inoculums suspension. The McFarland standard tubes were sealed with parafilm to prevent evaporation. McFarland standards then were stored in the dark at room temperature (22° to 25°C) (Wiegand et al., 2008).

3.7.1.2 Culture inoculation At least 3-5 well isolated colonies of the same morphological type were selected from the agar plate culture. The top of each colony was touched with a loop, and the growth was transferred into a tube containing 4 ml Tryptic soy broth (TSB) (Oxoid Ltd, Hampshire UK). The broth culture was either directly adjusted to the McFarland standards or by incubation at 35oC until it achieved or exceeded the turbidity of the 0.5 McFarland standards (usually 2-6 hours) (Figure 3.6).

A sterile cotton swab was dipped into the adjusted suspension. The swab was rotated several times pressed firmly on the inside wall of the tube above the fluid level. This removed excess inoculum from the swab.

The dried surface of a Mueller-Hinton agar (HiMedia, Mumbai, India) plate was inoculated by streaking the swab over the entire sterile agar surface. This procedure was repeated by streaking two more times, rotating the plate approximately 60o each time to ensure an even distribution of inoculum.

30


Figure 3.6 Comparison of culture suspension with 0.5 McFarland standard solutions.

3.7.1.3 Application of antibiotic discs to inoculated agar plates Antimicrobial discs (Table 3.1) were dispensed onto the surface of the inoculated agar plate. Each disc was pressed down individually to ensure complete contact with the agar surface. The disc placed in the agar surface was not closer than 24mm from center to center. A total of 6 discs were placed on one 150mm plate. Plates were then incubated at 37oC under 5% CO2 in incubator (Binder CB-150 CO2 Incubator, Chelmsford, UK). Table 3.1 Antibiotic discs used in this study No.

Antibiotics

Concentration

Brand

1.

Penicillin G

10µg

HiMedia, India

2.

Amoxicillin

10µg

Oxoid, UK

3.

Ciprofloxacin

5µg

Oxoid, UK

4.

Erythromycin

15µg

Oxoid, UK

5.

Gentamicin

30µg

HiMedia, India

6.

Ceftriaxone

30µg

Oxoid, UK

31


3.7.1.4 Reading plates and interpreting results After 24h of incubation, each plate was examined. The resulting zone of inhibition was uniformly circular with a confluent lawn of growth. The diameters of the zones of complete inhibition were measured, including the diameter of the disc. Zones were measured to the nearest whole millimeter, using regular scale. The zone margin was taken as the area showing no obvious, visible growth that could be detected with the unaided eye. The sizes of zones of inhibition were interpreted by referring to zone diameter interpretive standards (Tuohy et al., 2000) and organisms are reported as susceptible, intermediate or resistant to the antibiotics that have been tested.

32


3.7.2 Minimal Inhibitory Concentration (MIC) assay: microdilution technique

3.7.2.1 Preparation of antibiotic Stock solution Antibiotic stock solution was prepared using commercially available antimicrobial powders (with given potency). The amount needed and the diluents in which it can be dissolved were calculated by using the following formulas to determine the amount of antimicrobial powder for a standard solution:

Weight mg =

Volume ml × Concentration (μg/ml) Potency(μg/mg)

10ml antimicrobial stock solution was prepared at concentrations of 5000 μg/ml for each antibiotic. As microbial contamination is extremely rare, solutions those had been prepared aseptically and were sterilized by membrane filtration. Small volumes of the sterile stock solutions were dispensed into sterile vials; carefully sealed; and stored at −20°C. Vials were thawed as needed and used in the same day (EUCAST; Wiegand et al., 2008).

3.7.2.2 Preparation of antibiotic dilution range For broth microdilution, susceptibility panel in 96-well microtiter plates were prepared by dispensing 100μl antibiotic solution with the concentrations of 1024μg/ml into the first column wells and 50μl of sterile Luria-Bertani (LB) broth (Oxoid Ltd, Hampshire UK) into the rest wells by an 8-channel pipette. To make 1024μg/ml antibiotic solution at first column, 20.48μl stock antibiotic (5000 μg/ml) solution is added with 79.52μl distilled water. Then, the two-fold serial dilutions of antibiotic solutions were made by drawing up 50μl solution in the first column wells into the second column and then move on to the next column to achieve the final concentrations (Figure 3.7) (Wiegand et al., 2008).

33


Eight

antibiotics

(Gentamycin,

Chloramphenicol,

Azithromycin,

Ciprofloxacin,

Cefixime, Sulfamethoxazole, Ceftriaxone & Oxytetracycline) were used in eight row of microtiter plate (A-H) respectively to assay their Minimum Inhibitory Concentration (MIC) value for each of 21 isolates.

Figure 3.7 Serial dilution of antibiotic solution in a Microtiter plate using 8-channel pipette

3.7.2.3 Preparation of inoculum Bacterial inoculum was prepared by making a direct saline suspension of isolated colonies selected from a 24h Nutrient agar (NA) plate. The suspension was adjusted to achieve a turbidity equivalent to a 0.5 McFarland turbidity standard which resulted a suspension containing approximately 1.5 x 108 CFU/ml. (As described in 3.6.1.1) Prepared suspension was diluted in 1:150 using LB broth resulting in a suspension containing approximately 106 CFU/ml. The subsequent 1:2 dilution after adding 50μl suspension with 50μl antibiotic in each column except last column brought the final inoculum to 5 x 105 CFU/ml. Standard bacterial suspension (106 CFU/ml) was added with antibiotic (previously dispensed) in microtiter plate with in 15 min of preparation. Last column was kept free of inoculum and antibiotics as negative control (only media). 34


Previous column of last column was kept antibiotic free inoculation as positive control (EUCAST, 2003).

3.7.2.4 Incubation Inoculated plates were marked and sealed using parafilm. Plates were incubated at 37oC for 24h.

3.7.2.5 Measurement of turbidity After 24h incubation, each microtiter plates were measured using Microtiter plate reader (PowerWave-xs Microplate Spectrophotometer, BioTek, VT, US). Reading data was examined to determine MIC value (Figure 3.8).

Figure 3.8 MIC determinations based on the plate reader reading (PowerWave-XS, BioTek, VT, US)

35


3.8 Molecular Identification

3.8.1 Conventional Polymerase Chain Reaction (PCR) technique 3.8.1.1 DNA extraction 1. Biochemically positive pure bacterial samples were subcultured in Tryptone soya broth (TSB) (HiMedia, Mumbai, India) overnight at 37°C. 2. Overnight grown culture were dispensed in 1 ml volumes in sterile vials, 3. The turbidity of bacterial suspension was adjusted approximately to 1McFarland which equal approximately to 3× 108 CFU/ml. 4. The bacterial suspensions were centrifuged @13,000rpm for 15 min, 5. The supernatants were discarded and the pellets were washed three times using phosphate buffered saline (PBS), 6. Suspension of the pellet into 150µl of Nuclease free water (Qiagen GmbH, Hilden, Germany). 7. Bacterial suspension was subjected to boil at 100°C for 10 min. 8. Tubes were placed into ice immediately after boiling for another 10 min. 9. Tubes were centrifuged @13,000rpm for 10 min. 10. Supernatant were collected in a new 1.5ml tubes carefully to avoid any debris or pellets. 11. Extracted DNA was stored at -20oC.

36


3.8.1.2 PCR mastermix preparation Primer used for PCR reaction was as follows

Table 3.2 Primer sets used for PCR reaction Amplification

No

Primer

Type

Sequence

1.

d1

Forward

5′-CCITAYICITAYGAYGCIYTIGARCC-3′

size (Target)

480bp 2.

d2

Reverse

5′-ARRTARTAIGCRTGYTCCCAIACRTC-3′

PCR Mastermix was prepared as the formulation of following Table (3.3)

Table 3.3 Master mixture preparation for conventional PCR No. 1.

Reagent TaqMan® universal PCR Mastermix (ThermoFisher Scientific, MA, US)

Concentration

Volume/Reaction

2X

25µl

2.

Forward primer(sodA-d1)

10 pmol

2.5µl

3.

Reverse primer(sodA-d2)

10 pmol

2.5µl

4.

Nuclease free water (Qiagen GmbH,

10µl

Hilden, Germany)

Total volume of Master Mix

40µl

Extracted DNA template

10µl

Final volume of reaction

1X

37

50µl


3.8.1.3 DNA amplification Thermal cycling was performed in Stratagene Mx3005p (Stratagene California, CA, US) thermal cycler as following steps as (Table 3.4)

Table 3.4 Thermal cycling profile used for PCR reaction. No.

Steps

Definition

Temperature

Time

1.

First step

Initial Denaturation

95oC

3 min

2.

Second step

Denaturation

95oC

30 sec

3.

(30 cycles)

Annealing

37oC

60 sec

Elongation

72oC

60 sec

Final elongation

72oC

7 min

4. 5.

Third step

Amplified DNA product was stored at 4oC until gel electrophoresis

3.8.1.4 Preparation of 1.8% Agarose gel 1. 0.9gm agarose powder (ThermoFisher Scientific, MA, US) was added with 50ml TBE buffer in a conical flask. 2. The flask was heated for 2 min in a microwave oven. 3. Heated gel was cool to 60-70°C. 4. 2.5µl Ethidium Bromide from10mg/ml stock was added and mixed properly. 5. Gel tray was equipped with 25 well comb (1.5mm). 6. The mixture was poured on gel tray for cooling. 7. Proper safety precaution was taken during gel preparation due to the hazardous effect of Ethidium Bromide.

38


3.8.1.5 Gel electrophoresis 1. After gel formation agarose get was placed into gel running machine filled with TBE buffer. 2. PCR samples were stained with loading dye (2µl dye with 6µl PCR product). 3. Each DNA samples was loaded on individual well carefully using micropipette. 4. Electrophoresis was performed in Mupid®-One Electrophoresis System (Mupid Co., Ltd, Tokyo, Japan) for 30 min at 100V (Figure 3.9). 5. The gel was observed under UV light in gel-doc machine (Alphagram mini system, ProteinSimple, CA, US) and imaged using AlphaView software. Figure 3.9 Gel Electrophoresis performing in Mupid®-One Electrophoresis System.

39


3.8.2 Partial sequencing of sodA gene. 3.8.2.1 PCR amplification for partial sequencing. Target DNA (480bp) for partial sequencing of sodA gene was amplified using the same primer and same protocol as described in Conventional PCR technique (3.8.1).

3.8.2.2 Purification of amplified DNA Purification of amplified DNA was performed using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Protocol followed during purification process was as below: 1. 96–100% ethanol was added to Buffer PE to produce Ethanol-Buffer solution. 2. pH indicator I was added to Buffer PB at 1:250 volume. 3. PCR sample was added to Buffer PB a 1:5 volume. 4. A QIAquick spin column was placed in a 2 ml collection tube. 5. Sample-Buffer mixture was applied to the QIAquick column & centrifuged @13000rpm for 60 sec. 6. Flow-through was discarded & QIAquick column was placed back into the same tube. 7. 0.75 ml Buffer PE was added to the QIAquick column and centrifuged @13000rpm for 60 sec. 8. Discard flow-through & place the QIAquick column back in the same tube. 9. The column was centrifuged @13000rpm for 60 sec. 10. QIAquick column was placed in a clean 1.5 ml microcentrifuge tube. 11. 50μl of elution buffer (EB) was added to the center of the QIAquick membrane & centrifuge the column@ 13000rpm for 60 sec.

40


3.8.2.3 Quantification of DNA purity and concentration Each time after PCR reaction and purification process, amplified products were checked for purity and DNA concentration using Spectrophotometer (NanoDrop 2000c, ThermoFisher Scientific, DE, US). Procedure as follows: 1. 1µl Nuclease free water was loaded onto the lower measurement pedestal of NanoDrop 2000c and lowered the sampling arm. 2. Blank button was clicked using NanoDrop 2000c software (Figure 3.9) to measure and store the reference spectrum. 3. A 1µl DNA sample was applied onto the lower measurement pedestal and lowered the sampling arm. 4. DNA sample quality and quantity was measured and displayed as (Figure 3.10) after clicking Measure button with in 5 sec. 5. DNA concentration (ng/µl) and 260/280 value was recorded for each samples.

Figure 3.10 NanoDrop 2000c software, DNA concentration and purity measurement

41


3.8.2.4 Sequencing of target DNA using Sanger sequencing method Cycle sequencing was performed using BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, CA, US). Following procedure was maintained during purification process: 1. Completely thawed BigDye Terminator v3.1 Sequencing Kit and primer were store on ICE 2. Tubes were vortexed and then briefly centrifuged. 3. Reagents, template and primers were added according to following formula (Table 3.5) 4. The tubes were vortexed & centrifuged @1000g for 10 sec. 5. PCR run as following (Table 3.6) 6. Stratagene Mx3005p (Stratagene California, CA, US) was used for PCR thermal cycling (Figure 3.11).

Figure 3.11 Stratagene Mx3005p (Stratagene California, CA, US) thermal cycler used for Cycle sequencing of sodA amplified DNA.

42


Table 3.5 Cycle sequencing formula

No.

Reagents

Concentration

Volume/reaction (Forward reaction)

Volume/reaction (Reverse reaction)

2.5X

8µL

8µL

5X

4µL

4µL

1.

Ready Reaction Premix

2.

BigDye Sequencing Buffer

3.

Forward Primer

10 pmol

1µL

--

4.

Forward Primer

10 pmol

--

1µL

5.

Template DNA

(15)ng/µL

3µL

3µL

6.

Nuclease free water

4µL

4µL

20

20

Final Volume

1X

Table 3.6 Thermal cycle profile used for cycle sequencing No.

Steps

1.

First step

2.

Definition

Temperature

Time

Initial Denaturation

96oC

1 min

Denaturation

96oC

10 sec

Annealing

40oC

5 sec

Elongation

60oC

4 min

Second step 3. (40 cycles) 4.

43


3.8.2.5 Purification of Extended PCR products PCR extended product was purified using BigDye XTerminator purification kit (Applied Biosystems, CA, US) to sequester Cycle sequencing reaction components such as salt ions, unincorporated dye terminators and dNTPs. 1. Reaction tubes were centrifuged for 1 min, 10µl of each extended DNA was taken from 20µl of total Cycle sequenced product into a 96-well, v-bottom plate. 2. 45 µl of SAM solution was added to each well containing purified DNA. 3. XTerminator solution was vortexed at maximum speed for 10 sec to homogenize the solution. 4. 10 µL XTerminator solution was added using wide-bore pipette. 5. The plate was sealed using Adhesive films. 6. Plate was vortexed @1800rpm for 30 min. 7. Plate was spun @1000g for 2 min using swinging-bucket. 8. Adhesive film was removed and covered with the septa mat. 9. The plate was loaded into 3130 Genetic Analyzer (Applied Biosystems, CA, US) (Figure 3.12).

44


3.8.2.6 Sequence analysis: Capillary electrophoresis Capillary run of loaded samples into Applied Biosystems 3130 Genetic Analyzer (Figure 3.12) were formatted and launched using 3130 genetic analyzer data collection software 3.0 and Sequence analysis Plate editor.

Figure 3.12 Installation of sample and other required reagents into DNA sequence analyzer and starting the sequence analysis.

DNA sample loading in AB 3130 Genetic Analyzer

Sequencing running in AB 3130 Genetic Analyzer

45


3.8.2.7 Processing of obtained sequence and interpretation. After completing DNA sequencing analysis by capillary electrophoresis process of 3130 genetic analyzer (Applied Biosystems, CA, US) a chromatogram (.ab1) file had been generated in a predetermined location in hard disk of connected computer. Those files (.ab1) containing chromatogram information related to target-DNA sequence was used to extract final sequence. Final sequence determination and analysis was made as follows: 1. Chromatogram files (.ab1) were opened in Chromas V2.6.2 (Technelysium Pty Ltd) software (Figure 3.13) and low quality sequence read was excluded and amended if required. 2. Reverse DNA sequence was converted to reverse complementary and aligned with Forward DNA sequence of the same sample using BioEdit Sequence Alignment V7.2.5 (Hall, 1999)(Figure 3.14). 3. Aligned sequenced were assembled to generate consensus DNA sequence (contig) from forward and reverse sequence. 4. Final sequence was used as query sequence in Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) to find out the relevance of that sequence with the other sequence present in National Center for Biotechnology Information (NCBI) database. 5. By analyzing the BLAST search result final conclusion about the sample isolate was made. 6. Finally assembled sequence was converted into FASTA format and submitted on NCBI database.

46


Figure 3.13 Chromatogram view of genetic analyzer data collection software 3.0 output file (.ab1) in Chromas 2.6.2 software.

Figure 3.14 Sequence alignment and assembly in BioEdit Sequence Alignment editor 7.2.5 software.

47


3.8.3 Phylogenetic analysis of obtained sequences DNA sequence of present study isolates were compared with the similar DNA sequences previously published in Genbank database. Phylogenetic tree was constructed based on multiple sequence alignment using MEGA 6.0 (Tamura et al., 2013). The reliability of the branching orders was estimated by bootstrapping (1000 replicates). The phylogenic tree was rooted using partial sequence of Mycobacterium sp accession no. DQ822576.1. Gene sequences used in this study for Phylogenetic analysis is as follows

Table 3.7 Description of sodA partial sequences of different organism with their sources Organism/Isolates

Source

Genbank accession

CB-04

Commercial broiler cecum

KY386847

CB-08


KY386848

CB-09


Not published

CL-16

Commercial layer cloaca

Not published

CL-42

Commercial layer cloaca

Not published

BB-01

Broiler breeder cloaca

Not published

Enterococcus cecorum CIP

ATCC 43198

AJ387908.1

Enterococcus cecorum CE2 strain

Clinical case

CP010062.1

Enterococcus cecorum CE3 strain

Clinical case

CP010063.1

Enterococcus cecorum SA3 strain

Clinical case

CP010064.1

Enterococcus gallinarum

ATCC 49573

AJ387915.1

Enterococcus hirae

ATCC 8043

AJ387916.1

Enterococcus faecalis

Mouse

CP015410.1

Enterococcus mundtii

ATCC 43186

AJ387918.1

Enterococcus columbae

ATCC 51263

AJ387909.1

Mycobacterium tuberculosis

Human patient with suffering from sarcoidosis

DQ822576.1

103676 T strain

48

CHAPTER 4 – RESULTS

4.1 Bacterial isolates and identification results in Selective media A total of 136 samples were collected from Dhaka and Gazipur area consisted of three categories of source including Commercial Broiler (n=36), Commercial Layer (n=69), Broiler Breeder (n=31). Among them 42 isolates (Table 4.1) were selected as presumptive EC based on their growth characteristics in both selective media. In Kanamycin Aesculin Azide agar (KAA) culture growth characteristics of presumptive EC was as described in 3.5.1; colony with slightly gray, small and partially blackening of the media (Figure 4.1).

Figure 4.1 Growth characteristic of Isolated EC in Kanamycin aesculin azide (KAA) agar media.

Positive isolates in KAA agar culture were grown in Blood agar media supplemented with Colistin sulfate and Nalidixic acid. In Blood agar media presumptive EC culture was grown as described in 3.5.2; small, α-hemolytic colonies with greenish coloration of the media (Figure 4.2).

Results

Figure 4.2 Growth characteristic (α-hemolysis with slightly green pigmentation) of isolated EC in Blood agar (BA) media.

Among 136 samples 109 (80%) isolates were positive in KAA agar, from those only 42 (31%) were positive in Blood agar media. 42 isolates those showed positive growth characteristics in both KAA agar and Blood agar (Table 4.1) were presumed as EC and selected for biochemical tests.

Table 4.1 Growth result of samples in Selective media; CC: Cecal content, CS: Cloacal sample, M: Months, KAA+ve: Positive growth result in Kanamycin aesculin azide agar, BA+ve: Positive culture in Blood agar which was positive in KAA agar too. Age KAA+ve KAA+ve Sample No. of Sample source (n) (%) type samples variation Commercial broiler (CB) Commercial layer (CL)

BA+ve

BA+ve

Positive EC

(n)

(%)

(Presumptive)

CC

36

6-12M

18

50%

11

31%

11(31%)

CS

69

12-18M

68

99%

22

32%

22(32%)

CS

31

15-18M

23

74%

9

29%

9(29%)

136

6-18M

109

80%

42

31%

42(31%)

Broiler breeder (BB) Total

50

Results

4.2 Biochemical test results

4.2.1 Gram staining According to growth characteristics in selective culture media 42 isolates were subjected for gram staining. Among them 39 isolates showed gram positive criteria (Figure 4.3). The remaining 4 gram negative isolates were eliminated from the study and those 39 isolates were selected for further biochemical tests.

Figure 4.3 EC isolates under microscope after gram staining; Magnification: 1000x, Photo was taken using Olympus CX41, Olympus Corp., Tokyo, Japan.

51

Results

4.2.2 Catalase test A total of 39 Gram-positive bacteria isolates were examined for catalase enzyme production using 3% H2O2. From them 36 samples (Table 4.2) were catalase negative. Figure 4.4 Catalase test reaction (no bubble production) of EC isolates. Catalase (+) Salmonella spp

Catalase (-) No bubble formation

EC isolates

Ctalase(+) control

4.2.3 Esculin hydrolysis test result Test for Esculin hydrolysis performed in Bile esculin agar slant and all the tested 36 samples (Table 4.2) showed esculin hydrolysis by blackening the media (Figure 4.5). Figure 4.5 Esculin hydrolysis (Blackening of Bile esculin agar slant) by EC isolates.

A

B

C

D

E

F

G

H

A: Negative control, B-I: Esculin positive isolates.

52

I

Results

Table 4.2 Sequential biochemical test results of all selected isolates (Positive in selective media); CB: Commercial broiler, CL: Commercial layer, BB: Broiler breeder, NP: Test was not performed, P/N: (+)/(-) result, (*): Non-identical result for regular EC strains (Devriese et al., 1983). Sample ID

CB-2 CB-3 CB-4 CB-7 CB-8 CB-9 CB-15 CB-16 CB-17 CB-28 CB-29 CL-16 CL-18 CL-29 CL-30 CL-31 CL-32 CL-37 CL-38 CL-39 CL-40 CL-41 CL-42 CL-43 CL-52 CL-53 CL-54 CL-55 CL-56 CL-57 CL-63 CL-64 BB-1 BB-2 BB-3 BB-4 BB-20 BB-23 BB-24 Total (P/N)

Catalase

Esculin

Production hydrolysis

PYR

Selected as

Fermentation of

reaction

EC /non EC

Ribose

Raffinose

Lactose

Glycerol

(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (+)* (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (+)* (-) (-) (-) (-) (-) (-) (+)*

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) NP (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) NP (+) (+) (+) (+) (+) (+) NP

(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) NP (+)* (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (+)* NP (-) (-) (-) (-) (+)* (+)* NP

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (-)* (+) (+) (-)* (+) NP NP (+) (+) (+) (-)* (+) (+) (+) (+) (+) (-)* (+) (+) (+) NP NP (+) (+) (+) (+) NP NP NP

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) NP NP (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) NP NP (+) (+) (+) (-)* NP NP NP

(+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) (+) NP NP (+) (+) (+) (+) (+) (+) (-)* (+) (+) (+) (+) (+) (+) NP NP (+) (+) (+) (+) NP NP NP

(-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (+)* NP NP (-) (-) (+)* (-) (-) (-) (+)* (-) (-) (-) (-) (-) (-) NP NP (-) (-) (-) (+)* NP NP NP

EC EC EC EC EC EC EC EC EC EC EC EC EC EC EC Non-EC Non-EC EC EC EC EC EC EC EC EC EC EC EC EC EC Non-EC Non-EC EC EC EC EC Non-EC Non-EC Non-EC

3/36

36/0

4/32

28/4

31/1

31/1

4/28

32/7

53

Results

4.2.4 PYR Test Thirty six Esculin positive isolates were tested for the production of Pyrrolidonyl Arylamidase (PYR) enzyme in PYR culture media after addition of PYR reagent on suspected colonies as described (3.6.4). 32 Isolates (Table 4.2); those showed negative PYR test reaction (Figure 4.6) were confirmed as Enterococcus cecorum (EC).

Figure 4.6 PYR positive (non EC) and negative (EC) reaction showed in PYR agar growth after addition of PYR reagent.

PYR positive colony (Non-EC isolate)

PYR negative colony (EC isolate)

54

Results

4.2.5 Sugar fermentation profile of Enterococcus cecorum (EC) isolates. Fermentation result (Table 4.2) of four tested sugar were used to conform the isolates as EC. Among 32 isolates all of them except 2 were Ribose, Raffinose and Lactose positive (Figure 4.7). On the other hand 29 cultures were Glycerol negative. However all the 32 isolates were selected for further study like Antibiotic profiling and Molecular study.

Figure 4.7 Four types of fermentation test were performed for suspected isolates.

Control (-)ve (-)

(-)

(-)

(-)

(+)

ve

ve

ve

ve

ve

Glycerol Fermentation

Lactose Fermentation

(-) (-)

(+)

(+)

(-)

Lactose (+)ve

(-)

(-)

(-)

(+)

Raffinose Fermentation

Ribose Fermentation

55

(-)

Results

4.3 Antibiotic sensitivity assay result 4.3.1 Antibiotic resistance pattern in Kirby-Bauer disk diffusion In this study, 21 isolates including commercial broiler (n=10), commercial layer (n=7), and broiler breeder (n=4) were tested against six commercially available antibiotic disk like Ciprofloxacin (5µg), Amoxicillin (10µg), Erythromycin (15µg), Gentamicin (30µg), Ceftriaxone (30µg), and Penicillin-G (10µg). During the period of study, the sizes of zones of inhibition of every antibiotic disc were measured in millimeter and while those zones of inhibition compared with zone diameter interpretive standards from NCCLS 2000 and CLSI 2010 for Enterococcus, the isolates showed sensitive, intermediate or resistant to the antibiotic. Different isolates produce various sizes of inhibition zones (Figure 4.8; Table 4.3).

Figure 4.8 Zone of inhibition (ZI) of EC growth on Mueller-Hinton agar due to the antimicrobial effect of diffused antibiotics impregnated in each disk. Diameter of ZI (30mm)

56

Results

Among the tested isolates none of the bacteria were sensitive to all antibiotics whereas 100% showed resistance to at least two antibiotics. Of the tested isolates, 86% (18) were multidrug resistant (MDR) as defined in (Magiorakos et al., 2012) and showed resistance to three or more antibiotics from the six applied antibiotics. But none of the tested isolates were resistant to all antibiotics.

Table 4.3 Sensitivity of EC isolates against all six antibiotics; ZI: Zone of Inhibition, IC: Interpretive criteria, nz: no Zone of Inhibition. CB: Commercial broiler, CL: Commercial layer, BB: Broiler breeder, S: Sensitive, I: Intermediate, R: Resistance, Sample Ciprofloxacin Amoxicillin Erythromycin Gentamicin ID (5µg) (15µg) (15µg) (30µg)

ZI

ZI

(mm) IC

ZI

(mm) IC

ZI

(mm) IC

Ceftriaxone

Penicillin-G

(30µg)

(10µg)

ZI

(mm) IC

ZI

(mm) IC

(mm)

IC

CB-2

19

I

20

S

nz

R

17

S

19

S

nz

R

CB-3

8

R

20

S

nz

R

15

S

10

R

nz

R

CB-4

nz

R

17

S

nz

R

9

R

nz

R

nz

R

CB-7

nz

R

25

S

nz

R

14

I

15

R

nz

R

CB-8

nz

R

20

S

nz

R

10

R

08

R

nz

R

CB-9 CB-16

22 15

S R

17 18

S S

08 15

R I

12 17

R S

16 09

R R

09 nz

R R

CB-17

40

S

nz

R

20

I

30

S

10

R

nz

R

CB-28 CB-29

17 20

I I

17 nz

S R

15 25

I S

15 15

S S

14 10

R R

08 nz

R R

CL-16

23

S

15

R

17

I

nz

R

20

S

nz

R

CL-38

18

I

16

R

08

R

15

S

13

R

nz

R

CL-40

26

S

20

S

nz

R

15

S

15

R

nz

R

CL-42

21

S

10

R

nz

R

14

I

18

I

15

S

CL-52

40

S

nz

R

35

S

38

S

22

S

nz

R

CL-54 CL-55 BB-1 BB-2

28 20 23 21

S I S S

12 22 16 16

R S R R

10 20 nz 22

R I R I

20 nz 15 17

S R S S

17 14 17 16

R R R R

08 08 09 nz

R R R R

BB-3

20

I

30

S

nz

R

17

S

15

R

nz

R

14

R

16

I

15

S

17

R

10

R

20 I BB-4 Total R I S 5 7 9

10 0 11

12 7 2

57

5

2 14

17 1 3

20 0 1

Results

The resistance profile of MDR isolates ranged from 3 to 6 antibiotics is demonstrated in (Figure 4.9). In total of 21 tested isolates the highest percentages for MDR isolates were from Commercial broiler (CB). Among 18 MDR isolates 44% were from commercial broiler (n=8) which were resistant against three or more antibiotics including 3 isolates against three antibiotics, 3 isolates against four antibiotics, and 2 isolates against five antibiotics. Whereas none of the isolates except those 2 isolates from commercial broiler were resistance against five antibiotics. On the other hand 33% and 22% MDR isolates were from commercial layer and broiler breeder respectively.

Figure 4.9 Antibiotic resistance profile of three type isolates; CB: Commercial broiler, CL: Commercial Layer, BB: Broiler Breeder

10 9

No. of resistance isolates

8 7 6 5

BB CL

4

CB 3 2 1 0

2 resistances

3 resistances

4 resistances

58

5 resistances

Results

With regard to the prevalence of resistance to each antibiotic, Figure 4.10 shows the percentage of isolates resistant to each tested antibiotic. Among the isolates tested, the highest percentage of resistance was to Penicillin (95%), followed by Ceftriaxone (81%) and Erythromycin (57%) Amoxicillin (48%) Gentamicin (24%) Ciprofloxacin (24%). Whereas Gentamicin was most effective against the isolates, it was sensitive against 67% isolated EC.

Figure 4.10 Sensitivity profiles of each antibiotic against all isolates; S: % of Sensitive isolates, I: % of Intermediate isolates, R: % of Resistant isolates

100% 90% 80% 70% 60%

S

50%

I R

40% 30% 20% 10% 0%

59

Results

4.3.2 Antimicrobial susceptibility pattern of EC isolates in MIC test Antibiotic efficiency test of selected antibiotics against isolated samples were assayed using microdilution technique in microtiter plate. After full course incubation period microbial growth opacity was measured using Microtiter plate reader (PowerWave-XS Microplate Spectrophotometer) with the help Gen5 Microplate Reader and Imager Software V2.0 (Figure 4.10) at 600nm wave length. Opacity reading data were used to determine the MIC value as explained in Figure 3.7. The MIC distribution as well as MIC50 and MIC90 for the antimicrobial agents tested are shown in Table 4.4-A and Table 4.4-B.

Figure 4.11 Reading view of incubated microtiter plate used for (Minimal Inhibitory Concentration) MIC assay in (Gen5 Microplate Reader and Imager Software V2.0)

60

Results

Table 4.4-A Distribution of minimal inhibitory concentration (MICs: µg/ml) obtained by broth-dilution for 21 EC isolates (10-CB, 7-CL and 4-BB) from three types of sources; GEN: Gentamicin, CHL: Chloramphenicol, AZI: Azithromycin, CIP: Ciprofloxacin, CTX: Cefixime, SUL: Sulfamethoxazole, CFT: Ceftriaxone, OXY: Oxytetracycline, CB: Commercial broiler, CL: Commercial layer, BB: Broiler breeder, MIC50: Concentration of antibiotic (µg/ml) at which 50% of isolates were inhibited, MIC90: Concentration of antibiotic (µg/ml) at which 90% of isolates were inhibited. Antibiotics

GEN

CHL

AZI

CIP

CFX

SUL

CFT

OXY

Source

MIC50/ MIC90

CB

1

2

4

32

64

128

16

8/ 128

6

1

CL

16/ 128

3

1

BB

8/ 256

2

1

CB

32/ 512

4

3

CL

32/ 256

2

3

BB

128/ 256

CB

≥1024/ ≥1024

10

CL

≥1024/ ≥1024

7

BB

≥1024/ ≥1024

4

CB

32/ 64

1

CL

16/ 32

2

BB

1/ 64

CB

≥1024/ ≥1024

2

8

CL

≥1024/ ≥1024

3

4

BB

32/ ≥1024

CB

≥1024/ ≥1024

10

CL

≥1024/ ≥1024

7

BB

≥1024/ ≥1024

4

CB

8/ ≥1024

CL

256/ 512

BB

512/ ≥1024

CB

32/ 64

CL

32/ 256

3

BB

16/ 32

1

1

2 1

1

4

3

1

2

512

3

1

2 2 2

1

256

≥1024

8

1

3

2

1

1

2

2

2

4

1 2

1

3

1

1 2

2

61

6 3

2

1

1

1 1

3

1

Results

Table 4.4-B Overall distribution of minimal inhibitory concentration (MICs: µg/ml) obtained by broth-dilution for 21 EC against Eight antibiotics; GEN: Gentamicin, CHL: Chloramphenicol, AZI: Azithromycin, CIP: Ciprofloxacin, CTX: Cefixime, SUL: Sulfamethoxazole, CFT: Ceftriaxone, OXY: Oxytetracycline, MIC50: Concentration of antibiotic (µg/ml) at which 50% of isolates were inhibited, MIC90: Concentration of antibiotic (µg/ml) at which 90% of isolates were inhibited. AntiMIC50/ MIC90 biotics

GEN

8/ 128

CHL

32/ 256

AZI

≥1024/ ≥1024

CIP

32/ 64

CFX

≥1024/ ≥1024

SUL

≥1024/ ≥1024

CFT

256/ ≥1024

OXY

32/ 64

1

2

4

8

16

11

3

6

32

6

64

128

256

1

5

1

1

2

4

512

≥1024

2

21

2

3

1

4

6

4

1

2

5

14

21

1

2

4

4

4

62

2

1

10

1

3

1

1

4

4

Results

4.4 Conventional Polymerase Chain Reaction (PCR) result Conventional PCR technique was performed with sodA gene specific primers (Table 3.2) for 32 biochemically positive isolates. Among them 91% (n=29) had shown positive amplification in gel electrophoresis (Figure 4.12). Among the PCR positive samples 10, 15, and 4 isolates from Commercial broiler (CB), Commercial Layer (CL), and Broiler breeder (BB) respectively.

Figure 4.12 PCR positive isolates were observed as amplified DNA band near 480bp after the Gel electrophoresis of PCR product. Image of Gel electrophoresis was taken using gel-doc machine (Alphagram mini system, ProteinSimple, CA, US) and AlphaView software V2.1; bp: Size of nucleic acid in base pair.

900 bp 600 bp 480 bp 380 bp 300 bp 200 bp 100 bp

63

Results

4.5 Result of Partial sequencing of sodA gene 10 isolates positive in sodA gene specific PCR reaction were selected for partial sequencing of sodA gene. Purity and concentration of primarily amplified DNA were measured before and after purification process (3.8.2.2). Based on purity and concentration data (Table 4.5) of purified DNA eight samples were selected for cycle sequencing step. Purity and concentration measurement was done using NanoDrop 2000c Spectrophotometer (ThermoFisher Scientific, DE, US)

Table 4.5 Purity and concentration of primarily amplified DNA samples before and after DNA purification (Purification was done using QIAquick PCR Purification Kit-Qiagen, Hilden, Germany). Sample ID

DNA concentration (ng/µl) Before purification

Absorbance ratio 260nm/280nm

Absorbance ratio 260nm/230nm

After Before After Before After purification purification purification purification purification

CB-3

462.4

0.5

1.62

-0.6

1.03

-0.03

CB-4

462.3

10.5

1.62

2.03

1.03

-0.93

CB-8

465.6

9.6

1.62

2.11

1.04

-0.84

CB-9

471.0

9.6

1.62

2.1

1

-0.97

CL-16

532.3

12.2

1.64

2.19

1.06

-1.35

CL-29

511.9

13.8

1.64

2.11

1.06

-1.66

CL-42

750.6

8.4

1.61

2.61

0.88

-0.71

CL-52

174.9

4.5

1.63

2.56

0.97

-0.38

BB-1

474.0

6.0

1.61

2.55

1.01

-0.49

BB-3

466.6

2.2

1.62

5.07

1.02

-0.18

Average

477.16

7.73

1.62

2.273

1.01

-0.754

For absolutely pure DNA sample absorbance ratio between 260nm and 280nm should be around 1.8, lower value indicates contamination with protein and higher value means contamination with RNA. Absorbance ratio between 260nm and 230nm should be higher 64

Results

than the value of 260/280.

In this study 260/230 ratio decreases vigorously after

purification due to the presence of ethanol in sample DNA. Among the total of 8 successfully sequenced isolates 6 were identified as EC after aligning the sequence through Basic Local Alignment Search Tool (BLAST). It shows 90 to 99% sequence similarity among those sequences. Sequence was processed as described in 3.8.2.7. Final sequence details are given below.

Table 4.6 Partial sequence of studied EC isolates

Sample ID

Size of obtained

First 50 sequence (5´-3´)

sequence (bp)

CB-04

438

CB-08

467

CB-09

425

CL-16

416

CL-42

437

BB-01

491

ACAATCGATGAAGAAACAATGCATC TACATCATGAAAAACATCATAAAAC CCTTACGCTTATGATGCATTAGTACC TACAATCGATGAAGAAACAATGCAT CAATGCACTTACACCATGATAAACA CCACAACACTTATGTGACTAACTTA AGCCTTACATTGACGTGGAAACAAT GCACTTACACCATGATAAACACCAC CACTTANNCCNTGATAAACACCACA ACACTTATGTGACTAACTTAAACGC TCCGTATGCGTATGATGCGTTGGAG CCTTACATTGACGTGGAAACAATTG

65

Results

4.6 Phylogenetic analysis In this study, sodA partial sequences of 6 EC isolates (CB-04, CB-08, CB-09, CL-16, CL-42, BB-01) were compared with other sodA sequences of sequences previously published in Genbank as described in Table 3.7. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 2.15 is shown (Figure 4.13). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Saitou and Nei, 1987) and are in the units of the number of base substitutions per site. The analysis involved 15 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 275 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Phylogenetic analysis shows similarity of two isolates (CB-04, CB-08) from commercial broiler with clinical isolates from database. Other studied sequences have higher similarity with mouse isolates (E. faecalis accession no. CP015410.1) than the EC sequences.

66

Results

Figure 4.13 Phylogenetic tree of sodA gene sequence of present study isolates and Genbank sequence. The tree was constructed based on multiple sequence alignment using MEGA 6.0. Bootstrap value was used as 1000 for tree clustering. Number at the nodes represents the level of bootstrap support (%) based on the neighbor-joining analysis. The tree was rooted using partial sodA sequence of Mycobacterium sp accession no. DQ822576.1

Study Isolates

67

CHAPTER 5 - DISCUSSION

Worldwide prevalence and outbreak of Enterococcus cecorum (EC) is increasing day by day. Recent reports of several EC outbreaks in broiler farms especially in Europe and Americas increases its significance throughout the world (Devriese et al., 2002; Woo et al., 2004; Aziz and Barnes, 2007; Aziz and Barnes, 2009; Herdt et al., 2009; Stalker et al., 2010; Armour et al., 2011; Makrai et al., 2011; Robbins et al., 2012; Szeleszczuk et al., 2013; Aitchison et al., 2014; Zeshan et al., 2015; Borst et al., 2016; Packialakshmi et al., 2016). The objectives of current study were to isolate and characterize EC from the broiler breeder, commercial broiler, and commercial layer poultry in Bangladesh. And also to check their criteria against currently available antibiotics in Bangladesh.

136 samples were collected from three source including Commercial Broiler (n=36), Commercial Layer (n=69), Broiler Breeder (n=31). Among them 32 isolates have been found as EC including 11 from commercial broiler, 17 from commercial layer, and 4 from broiler breeder. That indicates the higher prevalence of EC in commercial broiler (35%) than the commercial layer (25%) and broiler breeder (13%). Prevalence of EC infection in commercial broiler have been reported previously (Jung and Rautenschlein, 2014) where 39% broiler chicken were infected with EC. So here in Bangladesh presence of EC in broiler is quite same. But in case of broiler breeder isolation rate of EC infection was much lower (13%) than (Kense and Landman, 2011) where prevalence rate was (47%).

In the current study 21 EC isolates including commercial broiler (n=10), commercial layer (n=7), and broiler breeder (n=4) isolates were tested with six antibiotics to check their resistance pattern. 86% (n=18) isolates were multidrug resistant (MDR) that includes commercial broiler (n=8; 80%), commercial layer (n=6; 86%), and broiler breeder (n=4; 100%) which is dangerously high for broiler and poultry industries in Bangladesh. MDR pattern of current study were quite similar with the other studies (Witte, 1998; Khan et al., 2005; Dolka et al., 2016) where high level of MDR isolates were observer too.

Discussion

Among 21 isolates high level of resistance were observed to penicillin-G (n=20; 95%), ceftriaxone (n=17; 81%), erythromycin (n=12; 57%), and amoxicillin (n=10; 48%). Whereas low level of resistance were observed against gentamicin (n=5; 24%), ciprofloxacin (n=5; 24%). Most isolates noted intermediate susceptibility to ciprofloxacin (n=7; 33%) and erythromycin (n=7; 33%) and none of the isolates were sensitive against all antibiotics. The result of current study was relevant with other studies (Khan et al., 2005) were higher resistance were demonstrated against erythromycin. Previous study of (Khan et al., 2005; Dolka et al., 2016) also showed the higher sensitivity of Enterococcus to gentamicin and ciprofloxacin which is compatible with this study. But increased level of penicillin-G and amoxicillin resistance among EC isolates in current study is really a matter to be worried about.

Minimal inhibitory concentrations (MICs) were determined by the broth microdilution method. A total of 21 isolates were tested against eight antibiotics including gentamicin, chloramphenicol, azithromycin, ciprofloxacin, cefixime, sulfamethoxazole, ceftriaxone, and oxytetracycline. Despite the lack of recommended breakpoints for enterococci, current study results suggest 100% prevalence of resistance to Azithromycin and Sulfamethoxazole. Higher MIC value also observed to ceftriaxone (MIC50/ MIC90 = ≥1024/ ≥1024), chloramphenicol (MIC50/ MIC90 = 32/ 256), and gentamicin (MIC50/ MIC90 = 8/ 128). Comparatively lower MIC value were found for ciprofloxacin (MIC 50/ MIC90 = 32/ 64) and oxytetracycline (MIC50/ MIC90 = 32/ 64). Comparing with other studies only 5 isolates against ciprofloxacin and 3 against ceftriaxone showed lower MIC value than the clinical breakpoint recommended for enterococci in the Clinical Laboratory Standards Institute (CLSI- 2010) (Dolka et al., 2016). All the other isolates showed higher MIC value against all antibiotics than the recommended breakpoint for enterococci.

In molecular characterization of 32 isolates 29 (91%) isolates were sodA gene specific PCR reaction. Phylogenetic analysis of 6 partially sequenced isolates were done MEGA6 (Tamura et al., 2013). Phylogenetic analysis shows similarity of two isolates (CB-04, CB-08) from commercial broiler with clinical isolates from database (Benson et al., 1993). Other studied sequences have higher similarity with mouse isolates (E. faecalis accession no. CP015410.1) than the EC sequences, which indicates sequence variation of sodA gene among the species level. 69

CHAPTER 6 – CONCLUSION

Enterococcus cecorum (EC) has been isolated from all type of study sources including Commercial Broiler, Commercial Layer, and Broiler Breeder. So it can be inferred that, Bangladeshi poultry has also been prevailed with commensal EC. According to the findings of present study the results can be concluded as follows: 

Documentation of the presence of EC in poultry for the first time in Bangladesh



EC prevalence rate in poultry is approximately 24% in Bangladesh



35% Commercial broiler, 25% commercial layer and 13% broiler breeder are infected by EC.



High level of multidrug resistant (MDR) isolates (86%) in poultry, where 100% isolates from broiler breeder are MDR.



Most of isolates show higher MIC value against Gentamycin, Chloramphenicol, Azithromycin, Cefixime, Ciprofloxacin, Sulfamethoxazole, Ceftriaxone, and Oxytetracycline as the recommended breakpoint determined by CLSI 2010.



Increased level of sequence variation in sodA gene among the isolates.

The final conclusion of this study can be said as the presence of EC infection in Bangladeshi broiler and their MDR profile is extremely crucial. So it is required to study their prevalence in clinical case like arthritis, osteomyelitis and spondylitic infection. Further research in Bangladesh is required to study more sources like other livestock sources and food chain. Study should be conducted to find their influence in human nosocomial infection as identified in other countries. The findings of this study will be helpful for those future studies of Enterococcus cecorum in Bangladesh

Reference

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APPENDICES

Appendix-A: Microbiological Media

A-01: Blood agar base (Oxoid, England) Ingredients

Amount (g/L)

Proteose peptone

15.0

Liver digest

2.5

Yeast extrac

5.0

Sodium chloride

5.0

Agar

12.0

pH

7.4 ± 0.2

A-02: Bile Esculin agar (HiMedia, India) Ingredients

Amount (g/L)

Peptic digest of animal tissue

5.0

Beef extract

3.0

Esculin

1.0

Bile salts

40.0

Ferric citrate

0.5

Agar

15.0

pH

6.6 ± 0.2

A-03: Brain Heart Infusion broth (Biotec, UK) Ingredients

Amount (g/L)

Brain infusion solids

12.5

Beef heart infusion solids

5.0

Peptocomplex

10.0

Glucose

2.0

Sodium Chloride

5.0

Disodium Hydrogen Phosphate

2.5

pH

7.4 ± 0.2

Appendices

A-04: Buffered Peptone Water (HiMedia, India) Ingredients

Amount (g/L)

Protease peptone

10.0

NaCl

5.0

Disodium phosphate

3.5

Monopotassium phosphate

1.5

pH

7.2 ± 0.2

A-05: Kanamycin Aesculin Azide agar (Oxoid, England) Ingredients

Amount (g/L)

Tryptone

18.8

Yeast extract

5.0

Sodium chloride

5.0

Sodium citrate

1.0

Aesculin

1.0

Ferric ammonium citrate

0.5

Sodium azide

0.15

Starch

0.6

Mix for Streptococci

0.6

Agar

10.0

Kanamycin sulphate

20.0

pH

7.0 ± 0.2

A-06: Luria Bertani broth (HiMedia, India) Ingredients

Amount (g/L)

Casein enzymic hydrolysate

10.0

Yeast extract

5.0

Sodium chloride

10.0

pH

7.5 ± 0.2

80

Appendices

A-07: Mueller-Hinton agar (Oxoid, England) Ingredients

Amount (g/L)

Beef extract

2.0

Acid hydrolysate of casein

17.5

Starch

1.5

Agar

13

pH

7.3 ± 0.1

A-08: Nutrient Broth (HiMedia, India) Ingredients

Amount (g/L)

Peptone

10.0

Sodium chloride

5.0

Disodium orthophosphate

9.0

Monopotassium orthophosphate

1.5

pH

8.5 ± 0.2

A-09: PYR broth (HiMedia, India) Ingredients

Amount (g/L)

Peptic digest of animal tissue

20.0

Dextrose

2.0

Sodium chloride

2.0

Disodium phosphate

0.4

Sodium carbonate

2.5

Chromogenic mixture

0.1

pH

7.8 ± 0.2

A-10: Tryptone Soya broth (Oxoid, England) Ingredients

Amount (g/L)

Pancreatic digest of casein

17.0

Papaic digest of soyabean meal

3.0

Sodium chloride

5.0

Dextrose

2.5

Dibasic potassium phosphate

2.5

pH

7.3 ± 0.2

81

Appendices

Appendix-B: Chemicals & Reagent

B-01: Phosphate buffered saline (PBS) Formula

Amount

KCl

0.2g

Na2HPO4

1.44g

NaCl

8.0g

KH2PO4

2.0g

Water

1L

B-02: 10 x TBE Formula

Amount

Tris-base

54.0g

Boric acid

27.5g

EDTA (0.5 M)

20ml

Water upto

500ml

B-03: Gel loading buffer (10X) Formula

Amount

Ficoll (20%)

800μl

EDTA (0.1M)

400μl

Bromophenol blue (0.25%)

10μl

SDS (1%)

200μl

Water

590ml

B-04: Ethidium bromide solution (Sigma, USA) Formula

Amount

Ethidium bromide

2.5g

Water

5ml

B-05: McFarland Solution Formula

Amount

BaCl2.2H2O (1.175%)

0.5ml

Water

99.5ml

82

Appendices

Appendix-C: Instruments Important instruments those were used in current study are given below Instruments

Company

Autoclave, Model No: SA-300V

STURDY, Taiwan

Centrifuge, Model:5804

Eppendorf, Germany

Class II Microbiological Safety Cabinet

Gelman Science, US

DNA Sequencer, Model: 3130 Genetic

Applied Biosystems, US

Analyzer Freezer (-80oc)

Angelentoni, UK

Gel Electrophoresis, Model: Mupid®-One

Mupid Co., Ltd , Japan

Gel Documentation, Model: Alphagram mini

ProteinSimple, US

Incubator, Model: CB-150

Binder, Germany

Light microscope, Model: CX41

Olympus Corp., Japan

Metter balance, Model: HR-200

METLER, Switzerland

Micropipettes

Labsystem, Finland

Microplate Spectrophotometer, Model:

BioTek, US

PowerWave-xs Oven Drier, Model: ED-115

Binder, Germany

PH Meter, Model: Mp220

Hach, US

Refrigerator (-20oC), Model: 360LTR

Samsung, South Korea

Spectrophotometer, Model: NanoDrop 2000c

ThermoFisher Scientific, US

Thermocycler, Model: Mx3005p

Stratagene California, US

Water Bath, Model: Sub6

England

83