Isolation and antibiogram of pathogenic bacteria (Escherichia coli, Salmonella sp.& Staphylococcus sp.) from different kinds of cheese available in market of Chattogram in Bangladesh.

Nusrat Sultana

Roll No. 0117/10

Registration No. 00413

Session: 2017-2018 (July-December)

A thesis submitted in the partial fulfillment of the requirements for the degree of
Master of Science in Food Chemistry and Quality Assurance

Department of Applied Chemistry and Chemical Technology
Faculty of Food Science and Technology

Chattogram Veterinary and Animal Sciences University
Chattogram-4225, Bangladesh

December, 2018

Authorization

I hereby declare that I am the sole author of the thesis. I also authorize the Chittagong Veterinary and Animal Sciences University (CVASU) to lend this thesis to other institutions or individuals for the purpose of scholarly research. I further authorize the CVASU to reproduce the thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research.

I, the undersigned, and author of this work, declare that the electronic copy of this thesis provided to the CVASU Library, is an accurate copy of the print thesis submitted, within the limits of the technology available.

Nusrat sultana The Author December, 2018

Isolation and antibiogram of pathogenic bacteria (Escherichia coli, Salmonella sp. & Staphylococcus sp.) from different kinds of cheese available in market of Chattogram in Bangladesh.

Nusrat sultana

Roll No. 0117/10

Registration No. 413

Session: 2017-2018 (July-December)

This is to certify that we have examined the above Master’s thesis and have found
that  is complete and satisfactory in all respects, and that all revisions required by the thesis examination committee will be addressed

--------------------------------------------------------

(Md. Fahad bin Quader) Supervisor

-------------------------------------------------------- (Md. Fahad bin Quader)

Chairman of the Examination Committee

Department of Applied Chemistry and Chemical Technology

Faculty of Food Science and Technology 
Chattogram Veterinary and Animal Sciences University Khulshi, Chattogram-4225, Bangladesh

December, 2018

DEDICATED TO MY

RESPECTED AND BELOVED PARENTS, SISTER AND TEACHERS

Acknowledgements

The author remembers the Almighty “ALLAH”, the omnipotent, omnipresent and omniscient who empowers the author to complete the work of this dimension and the manuscript successfully.

From the core of my heart, I would like to expresses the first and foremost heartiest appreciation,  deepest  sense  of  gratitude  and  indebtedness  to  my  respectable  and honorable research supervisor Md. Fahad bin Quader (Assistant Professor and Dept. Head, Department of Applied Chemistry and Chemical Technology, Faculty of food science & Technology, CVASU). Without his guidance it would not be possible for me to complete the research and then write up the dissertation successfully.

I also gratefully acknowledge the support, cooperation  and  encouragement  received during my MS program from all technical and non-technical staffs of the Department of Applied Chemistry and Chemical Technology, CVASU. I am also grateful to Dr. Sharmin Akter (Assistant Professor, Dept. of Veterinary Medicine and Surgery) for her valuable suggestions and cooperation during the thesis writing.

I wish to convey my gratefulness to Dr. Inkeyas Uddin, Scientific Officer, Poultry Research and Training Center (PRTC) for providing all the lab facilities to carry out my research work and special thanks to other staffs of PRTC for their technical support and cooperation during laboratory evaluation of samples. I sincerely thank to the University Grants Commission for giving me a research grant to accomplish my research work.

I  am  ever  indebted  to  my beloved  parents  and  friends  for  their  immense  sacrifice, blessing and encouragement.

The Author

December
2018

Table of Contents

List of contents Authorization……………………………………………………………………………...II Signature page……………………………………………………………………………III Acknowledgements……………………………………………………………………….V Table of Contents…………………………………………………………………….VI-IX
List of tables………………………………………………………………………………X List of figures………………………………………………………………………..XI-XII Acronyms used…………………………………………………………………………XIII Abstract………………………………………………………………………………...XIV

CHAPTER I………………………………………………………………………………1

INTRODUCTION………………………………………………………………………...1

1.1      Aim and objectives of the study…………………………………………………4

1.1.1      Aim………………………………………………………………….………4

1.1.2      Objectives…………………………………………………………………………4

CHAPTER- II………...................................................................................................................5

REVIEW OF LITERATURE……………….…………………………………………….5

2.1. Enterobacteriaceae…………………………………...……………………………..7

2.1.1.     Escherichia coli…………………………………………………………..…7

2.1.2. Diversities of pathogenic E. coli ………………..…………………………….7

2.1.3.     Enterotoxins of E. coli………………………………………………...……8

2.1.4.     Virulence factors………………………………………………………........8

2.1.5. Shiga toxin-producing/ Enterohaemorrhagic E. coli (STEC/EHEC)……...…11

2.1.6. Nomenclature………………………………………………………………....11

2.1.7. Pathogenesis…………………………………………………………………..11

2.1.8. Detection…………………………………………………………………...…12

2.1.9. Resistant genes of E. coli……………………………………………………..13

2.2.     Staphylococcus sp……………………………………………………………...14

2.2.1. Morphology of Staphylococcus sp………………………………………………....14

2.2.2. Staphylococcal surface protein……………………………………………….15

2.2.3. Pathogenesis…………………………………………………………………..15

2.2.4.     Human infections by Staphylococcus sp……………………………………....16

2.2.5. Symptoms ……………………………...………………………………………….17

2.2.6. Cheese and dairy products implicated with Staphylococcus sp……………….17

2.2.7. Vehicles of transmission………………………………………………….......18

2.2.8. Antibiotic susceptibility testing of Staphylococcus sp…………………………..18

2.2.9 Public health and economic importance………………………………………19

2.3. Salmonella sp……………………………………………………………………..20

2.3.1 Morphology of Salmonella sp…………………………………………………….…20

2.3.2. Transmission vehicles……………………………………………………...…20

2.3.3. Cheese borne Salmonellosis………………………………………………….21

2.3.4. Prevalence and varying factor of presence in cheese………………………...22

CHAPTER-III………………………………………………………………………..…23

MATERIALS AND METHODS………………………………………………………...23

3.1 Study population and sample collection………………………………………...…23

3.2 Experimental design…………………………………………………………....24-25

3.3 Physicochemical Parameters Determination……………………………………....26

3.3.1 Moisture determination…………………………………………………….… 26

3.3.2 Determination of pH………………………………………………………..…26

3.3.3 Titratable Acidity…………………………………………………………….. 27

3.4 Sample preparation……………………………………………………………….. 27

3.5 APC (Aerobic Plate Count)…………………………………………………… 27-28

3.6 Isolation and identification of Escherichia coli…………………………………....29

3.6.1 Biochemical tests performed………………………………………………......29

3.6.1.1. Indole test…………………………………………………………………..29

3.6.1.2. Carbohydrate fermentation test…………………………………………….29

3.6.1.3 Preservation of the culture…………………………………………………..29

3.7 Isolation and identification of Staphylococcus sp………………………………...30

VII

3.7.1 Preparation of Horse plasma for Coagulase test……………………………....30

3.7.1.1 Tube coagulase test……………………………………………………….…30

3.7.1.2. Tube catalase test…………………………………………………………...30

3.7.1.3 Preservation of the culture……………………………………………….….31

3.8 Isolation of Salmonella sp…………………………………………………………31

3.8.1 Identification of Salmonella by Biochemical characterization……………......31

3.8.1.1 Triple sugar iron (TSI) agar test…………………………………………….31

3.9 Antimicrobial susceptibility profile testing of bacterial isolates (Staphylococcus sp.
, Escherichia coli and Salmonella sp.)………………………………………………....33

3.9.1 Disk-diffusion procedure…………………………………………………..33-36

3.10 Detection of resistant and virulent genes of E. coli by Polymerase chain
Reaction ……………………………………………………………………………….37

3.10.1 Boiling method………………………………………………………………37

3.10.2 Preparation of working solution of primer…………………………………..39

3.10.3 Detection of resistant and virulent genes……………………………………39

3.10.4 Visualization of PCR Product……………………………………………….42

3.11 Statistical Analysis……………………………………………………………….42

CHAPTER IV..................................................................................................................43

RESULTS………………………………………………………………………………..43

4.1 Confirmation of E. coli by cultural characteristics and biochemical analysis……..45

4.2 Confirmation of Staphylococcus sp. and Salmonella sp. by cultural characteristics
and  biochemical analysis:……………………………………………………………..47

4.3 Confirmation of resistance pattern of E. coli, Staphylococcus sp. and Salmonella
sp. by CS tests……………………………………………………………………...….48

4.4 Confirmation of E. coli resistant and virulent genes by PCR………………….51-53

CHAPTER V…………………………………………………………………………....54

DISCUSSIONS……………………………………………………………………....54-56

CHAPTER VI...................................................................................................................57

CONCLUSIONS………………………………………………………………………...57

CHAPTER VII…………………………………………………………………………..58

RECOMMENDATIONS……………………………………………………………..58-59

CHAPTER VIII……………………………………………………………………...….60

REFERENCES …………………………………………………...………………….60-71

APPENDIX…………………………………………………………………………..72-75

BRIEF BIOGRAPHY

LIST OF TABLES

NO OF

TABLES	NAME OF TABLES	PAGE

NO	
1.	Intestinal pathogenic E. coli	9	
2.	Extra-intestinal pathogenic E. coli (ExPEC)	10	
3.	Panel of antibiotics used, their concentrations and Zone

diameter interpretative standards for E. coli and Salmonella sp. (CLSI, 2007)	34	
4.	Panel of antibiotics used, their concentrations and Zone

diameter interpretative standards for Staphylococcus sp. (CLSI, 2007)	35	
5.	Oligonucleotide primers used in PCR to detect tetracycline

resistant genes	39	
6.	Oligonucleotide primers used in PCR to detect virulent genes	40	
7	Contents of each reaction mixture (25 µl) of PCR	40	
8.	Specific Temperature and time for resistant (Tet A, Tet B &

Tet C) and virulent genes (Stx1, Stx2) were used in PCR of

E. coli	41	
9.	APC (Aerobic plate count) of collected cheese samples in

cfu/ml.	43	
10.	Summary statistics for moisture, pH and acidity	44	
11.	Frequency distribution for bacteria isolated from cheese	44	
12.	Overall prevalence of Escherichia coli.		
13.	Overall prevalence of Staphylococcus sp.	47	
14.	Prevalence of resistant genes of E. coli in cheese sample	52	

X

LIST OF FIGURES

NO OF

FIGURES	
NAME OF FIGURES	PAGE

NO	
1	Interaction of pathogenic E. coli with Eukaryotic cells.	10	
2	Geographical locations of the study area (CVASU).	23	
3	Serial dilution used in APC.	28	

4	Steps of Antimicrobial susceptibility test of E. coli,

Staphylococcus sp. and Salmonella sp. A. Streaking on the Mueller- Hinton Agar, B. Antibiotic disk, C. Inserting of Antibiotic disk onto the agar plate, D. Antibiotic resistant.	

36	

5	DNA extraction protocol for E. coli A. Taking a loopful of

colony from Blood agar B. Dissolved in autoclaved deionized water C. Vortexing of sample D. Pinching the head of Eppendorf, E. Placing into the ice F. tube placing into the water bath.	

38	
6	Line diagram of APC.	43	

7	Pie diagram of bacteria isolated from cheese.	
45	

8	A. Whitish color colony on Blood agar (E. coli) B. Pinkish color

colony on McConkey agar (E. coli) C. Metallic sheen on EMB agar (E. coli) D. Red color Upper ring in indole test (E. coli) E. Color change and gas production in Carbohydrate fermentation test (E. coli).	

46	

9	A. Yellowish colony on MSA for Staphylococcus sp. B. Heavy coagulation in tube coagulase test in Staphylococcus sp.	

48	

10	A. Antimicrobial sensitivity test for E. coli. B. Antimicrobial

sensitivity test for   Staphylococcus sp. C. Antimicrobial	
49	

XI

	sensitivity test for Salmonella sp.		

11	Frequencies of E. coli isolates showing resistant (R),

intermediately resistant (I) and sensitive (S) to the antimicrobials tested.	

50	

12	Frequencies of Staphylococcal isolates showing resistant (R),

intermediately resistant (I) and sensitive (S) to the antimicrobials tested.	

50	

13	Frequencies of Salmonella isolate showing resistant (R),

intermediately resistant (I) and sensitive (S) to the antimicrobials tested.	

51	
14	Results of PCR assay for Tetracycline resistant genes of E. coli.	53	

15	
Results of PCR assay for Stx-1 & Stx-2 genes of E. coli.	
53	

XII

LIST OF ACRONYMS AND SYMBOLS USED

Abbreviation and symbols     Elaboration	
%	Percent	
°C	Degree Celsius	
AMR	Antimicrobial Resistance	
BHI	Brain Heart Infusion	
bp	Base Pair	
BPW	Buffered Peptone Water	
CDC	Center for Disease Control and Prevention	
CFU	Colony Forming Unit	
CI	Confidence Interval	
CLSI	Clinical and Laboratory Standards Institute	
CS	Culture Sensitivity	
CVASU	Chittagong Veterinary and Animal
Sciences University	
DAEC	Diffusely Adherent E. coli	
DNA	De-oxy Ribonucleic Acid	
EAggEC	Enteroaggregating E. coli	
E. Coli	Escherichia coli	
EHEC	Enterohemorrhagic E. coli	
EMB	Eosin Methylene Blue	
ETEC	Enterotoxigenic E. coli	
GPS	Global Positioning System	
HC	Hemorrhagic Colitis	
HUS	Hemorrhagic Uremic Syndrome	
hrs	hours	
Kb	Kilo Base	
LPS	Lipopolysaccharide	
LT	Heat Labile Toxin	
μg	Microgram	
μl	Microliter	
MDR	Multi Drug Resistance	
mm	Millimeter	
ml	Milliliter	
OR	Odds Ration	
PBP	Penicillin Binding Protein	
PCR	Polymerase Chain Reaction	
PRTC	Poultry Research and Training Center	
RNA	Ribonucleic Acid	
rpm	Rotation Per Minute	
ST	Heat Stable Toxin	
TAE	Tris Acetate EDTA	
UTI	Urinary Tract Infection	
WHO	World Health Organization	

XIII

ABSTRACT

Cheese is a solid, fresh or ripened product obtained by adding the enzyme rennet in the presence of lactic acid produced by the adventitious microorganisms that causes coagulation and whey separation of milk, cream or partly skimmed milk, buttermilk. Cheeses of different types especially soft and semi-soft are suspected to the presence of pathogenic E. coli, Staphylococcus sp., Salmonella sp. etc which are harmful for public health. The purpose of the current research was to investigate the qualities of cheese collected from super-shops of Chattogram. A total of 15 cheese samples were collected from three super-shops of Chattogram named as shop-A, shop-B and shop-C. Among 15 cheese samples, the S3  sample collected from shop-B had the highest bacterial load
(1.80×107 cfu/ml) than those in shop-A and shop-C. Isolation of pathogenic E. coli, Staphylococcus sp., Salmonella sp. was done by using selective media such as EMB agar, MSA, XLD agar respectively. Four out of the total 15 cheese samples were found to be contaminated with E. coli (26.67%) while 1 sample was contaminated with Salmonella sp. (6.67%) & ten were detected as contaminated with Staphylococcus sp. (67%). The antibiotic susceptibility test was done by Kirby-Bauer method to detect susceptibility to ampicillin, amoxycilin, gentamicin, ceftriaxone, ciprofloxacin and tetracycline. E. coli, Staphylococcus sp., & Salmonella sp. showed almost 100% resistant behavior to ampicillin,  amoxycilin,  tetracycline  &  50%  sensitivity of  E.  coli  to  gentamicin  and ciprofloxacin while for Staphylococcus sp. sensitivity to gentamicin, ceftriaxone, ciprofloxacin was 27.27%, 90.91%, 18.18% respectively. The CS test results were highly significant for ceftriaxone (p=0.003; i.e. p<0.05). The screening for shiga-toxin strains in E. coli isolates was based on the detection of the two genes stx1 & stx2 and tetracycline resistant strains of E. coli were detected for Tet A, Tet B & Tet C genes by polymerase chain reaction (PCR). The prevalence of stx1 was found 25% while for stx2 50%. The prevalence of resistant genes specific to Tet A, Tet B of E. coli for cheese samples was
100%  (0.40-1.00 % CI) in 95% CI whereas for Tet C, prevalence was 50% (0.07-0.93 % CI) in 95% CI. In conclusion, the risk of pathogenic bacteria is higher in cheese if safety measures not maintained & could pose a massive health risk.

Key words: Cheese, Pathogen, antibiotic, resistant, CS, PCR, Shiga toxin.

XIV