iv. results - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/34415/11/11_chapter4.pdf · as...

IV. RESULTS

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4.1. Characterization and role of class 1 integron in antibiotic resistance of Salmonella

4.1.1. Detection of Salmonella spp. from seafood

The three genus specific primers were used for the identification of 65 strains of

Salmonella isolated from seafood by PCR. A product of 152, 284, and 216 bp (Figs. 10A, B

and C) was obtained by using primer hns, invA and hilA respectively from all the 65 strains of

tested Salmonella.

4.1.2. Antibiogram analysis of different serotypes for the detection of class 1 integron

Forty Salmonella isolates (17 S. Weltevreden, eight S. Paratyphi C, two S. Anatum,

eight S. Oslo, three S. Typhimurium and two S. Newport) used in the detection of class 1

integron were resistant to at least one antibiotic, and 43.50 % of the isolates were resistant to

two antibiotics. Twenty five percentages of our isolates were multidrug resistant, that is,

resistant to more than two of all the tested antibiotics. In the case of human adapted serovar S.

Paratyphi C, two of eight (25 %) tested isolates were multidrug resistant. In the case of

nontyphoidal serovars of Salmonella, four of the 17 tested isolates of S. Weltevreden (23.5 %)

were multidrug resistant. One isolate of S. Weltevreden (SW37) was resistant to four

antibiotics, while another one (SW9) was resistant to six antibiotics. Similarly, two (SO9 and

SO11) of eight tested isolates of S. Oslo (25 %) and two isolates of S. Newport (SN36 and

SN33) were resistant to multiple antibiotics (Table 12).

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Fig. 10. (A) PCR amplification of invA gene (284 bp), Lane M: 100 bp DNA Ladder

(GeneiTM

, Merck Bioscience, Bangalore), Lane 1: Positive control (ATCC 14028); Lane

2: Negative control; Lanes 3-8: Samples positive for Salmonella spp.; Lane 9: Sample

negative for Salmonella spp. (B) PCR amplification of hns gene (152 bp). Lane M: 100 bp

DNA Ladder (GeneiTM

, Merck Bioscience, Bangalore); Lane 1: Positive control (ATCC

14028); Lane 2: Negative control; Lanes 3-8: Samples positive for Salmonella spp.; Lane

9: Sample negative for Salmonella spp. (C) PCR amplification of hilA gene (216 bp).

Lane M: 100 bp DNA Ladder (GeneiTM

, Merck Bioscience, Bangalore); Lane1: Positive

control (ATCC 14028); Lane 2: Negative control; Lanes 3-8: Samples positive for

Salmonella spp.; Lane 9: Sample negative for Salmonella spp.

C B A

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Table 12. Antimicrobial resistance patterns among different Salmonella serovars

Resistance

pattern

Total SW

n = 17

SP

n = 8

SAN

n = 2

SO

n = 8

ST

n = 3

SN

n = 2

E 13 10 1 1 1 - -

AE 10 1 5 1 1 2 -

ET 6 1 - - 4 1 -

CoE 1 1 - - - - -

AEK 2 - 2 - - - -

AET 2 2 - - - - -

ACET 2 1 - - 1 - -

ACoCfENaC 4 1 - - 1 - 2

SW, S. Weltevreden; SP, S. Paratyphi C; SAN, S. Annatum; SO, S. Oslo; ST, S. Typhimurium;

SN, S. Newport

T, tetracycline; A, ampicillin; K, kanamycin; Na, nalidixic acid; E, erythromycin; Cf,

ciprofloxacin; Co, co-trimoxazole; C, chloramphenicol

4.1.3. Integron PCR analysis of the different serovars

Presence of six tetracycline-resistant genes, viz. tetA, tetB, tetC, tetD, tetE and tetG,

and one chloramphenicol resistant gene catA1 was tested using specific primers (Table 4). For

the isolates used in this study, the phenotypic expression of resistance in antibiogram was

always accompanied by the presence of the corresponding gene encoding for the particular

resistance. The tetracycline resistant isolates of S. Weltevreden contained tetA gene that

encodes for membrane associated efflux protein containing 12 transmembrane segments. In

this study, the tetA gene was located on the plasmid in all the tested isolates. The 817 bp

sequence obtained revealed 98 % sequence identity with the existing sequences of tetA, and as

this is the first report of tetA gene in the seafood isolates of S. Weltevreden (SW9), the

sequence has been submitted to NCBI GenBank and assigned accession number (EF535836)

(Fig. 11). Some of the isolates contained other tetracycline resistant genes such as tetB and

Results

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tetG (SW9 and SN36 respectively). The 560 bp region of the tetB gene of SW9 has been

sequenced and submitted to the NCBI GenBank and assigned GenBank accession number

(JN676152) (Fig. 12). None of the isolates contained tetC, tetD and tetE genes.

In this study, one strain (SW9) that showed resistance to chloramphenicol was positive

for the presence of catA1 gene by PCR. The 508 bp sequence of S. Weltevreden was obtained

from the amplicon revealed 100 % sequence identity with the existing sequences of catA1 in

the GenBank. As this is the first report of catA1 gene in seafood associated S. Weltevreden,

the sequence has been submitted to NCBI GenBank and assigned accession number

(EF523685) (Fig. 13). However, some strains which were chloramphenicol sensitive also

possessed the catA1 gene as confirmed by PCR (Table 13). The sequence obtained from the

sensitive strain (SW30) has also been submitted to the NCBI GenBank and assigned accession

number (JN685592) (Fig. 14). The primers cat F2/R2 used for the amplification of promoter

region of catA1 gene amplified a 528 bp product when DNA of SW9 was used and no

amplification was observed in SW30 (Fig. 15). Presence of class 1 integrons in multidrug

resistant seafood isolates were screened using different primers (Table 4). One strain of S.

Weltevreden (SW9) and two strains of S. Newport (SN36 and SN33) were positive for

integron. PCR analysis of class 1 integron revealed the presence of an amplicon of 800 bp in

SW9 and an amplicon of 1200 bp in SN36 and SN33 when primers int1F (5ꞌCS) and int1B

(3ꞌCS) were used (Fig. 16A), suggesting the presence of class 1 integron. Further, using

primers qacEΔ1F and sulB, a 800 bp PCR products was obtained (Fig. 16B) in all the three

isolates (SW9, SN36 and SN33). The combination of primer int1F (5ꞌCS) and sulB showed the

presence of 1597 bp PCR product in SW9 and 2055 bp product in case of SN36 and SN33

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(Fig. 16C). There was an excellent correlation between the presence of gene cassettes and the

corresponding antibiotic resistance phenotype of the isolates. Integron carrying isolates

showed resistance against co-trimoxazole (trimethoprim + sulfamethoxazole). S. Weltevreden

(SW9) contained a dihydrofolate reductase gene (dhfrA7) and a dihydropteroate synthetase

gene along with the usual quaternary ammonium compound resistance gene. As this is the first

report on the presence of integron gene in seafood associated S. Weltevreden (SW9), the

sequence has been submitted to NCBI GenBank and assigned accession number (HM769861)

(Fig. 17). Similarly, the presence of dihydrofolate reductase type 1, OrfC, quaternary

ammonium compound resistance and sulphonamide resistance gene associated with the

integron was identified in two seafood isolates of S. Newport (SN36 and SN33), and the

sequence has been submitted to NCBI GenBank and assigned accession numbers (JF800673

and JF800674) (Fig. 18). The obtained sequence data reveal that the genes are arranged in

contiguous manner and based on these sequence a map of integron of S. Weltevreden (SW9)

(Fig. 19A) and S. Newport (SN36) were drawn (Fig. 19B).

Results

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1 ATG GGG TTT CTC TTA TAT CCG GGC GGA TCG TGC CGG GCA TCA CCG GGG CGA CTG GGC GTA

M G F L L Y P G G S C R A S P G R L G V 61 GCT GCG CTA TAT GGC GAT ATC ACT GAT GCG ATG AGC GCG CGC GGC ACT CGG CTT CAT GAG

A A L Y G D I T D A M S A R G T R L H E 121 CGC TGT TTC GGG TTC GGG ATG GTC GCG GGA CCT GTG CTC GGT GGG CTG ATG GGC GGT TTC

R C F G F G M V A G P V L G G L M G G F 181 TCC CCC CAC GCT CCG TTC TTC GCC GCG GCA GCC TTG AAC GGC CTC AAT TTC CTG ACG GGC

S P H A P F F A A A A L N G L N F L T G 241 TGT TTC CTT TTG CCG GAG TCG CAC AAA GGC GAA CGC CGG CCG TTA CGC CGG GAG GCT CTC

C F L L P E S H K G E R R P L R R E A L 301 AAC CCG CTC GCT TCG TTC CGG TGG GCC CGG GGC ATG ACC GTC GTC GCC GCC CTG ATG GCG

N P L A S F R W A R G M T V V A A L M A 361 GTC TTC TTC ATC ATG CAA CTT GTC GGA CAG GTG CCG GCC GCG CTT TGG GTC ATT TTC GGC

V F F I M Q L V G Q V P A A L W V I F G 421 GAG GAT CGC TTT CAC TGG GAC GCG ACC ACG ATC GGC ATT TCG CTT GCC GCA TTT GGC ATT

E D R F H W D A T T I G I S L A A F G I 481 CTG CAT TCA CTC GCC CAG GCA ATG ATC ACC GCC CCT GTA GCC GCC CGG CTC GGC GAA AGG

L H S L A Q A M I T A P V A A R L G E R 541 CGG GCA CTC ATG CTC GGA ATG ATT GCC GAC GGC ACA GGC TAC ATC CTG CTT GCC TTC GCG

R A L M L G M I A D G T G Y I L L A F A 601 ACA CGG GGA TGG ATG GCG TTC CCG ATC ATG GTC CTG CTT GCT TCG GGT GGC ATC GGA ATG

T R G W M A F P I M V L L A S G G I G M 661 CCG GCG CTG CAA GCA ATG TTG TCC AGG CAG GTG GAT GAG GAA CGT CAG GGG CAG CTG CAA

P A L Q A M L S R Q V D E E R Q G Q L Q

721 GGC TCA CTG GCG GCG CTC ACC AGC CTG ACC TCG ATC GTC GGA CCC CTC CTC TTC ACG GCG

G S L A A L T S L T S I V G P L L F T A 781 ATC TAT GCG GCT TCT ATA ACA ACG TGG AAC GGG TGG

I Y A A S I T T W N G W

Fig. 11. Nucleotide sequence of tetracycline resistance class A (tetA) gene of tetracycline

resistant S. Weltevreden strain SW9

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1 GTG CTG TTG TTG TCA TTA ATA GGC GCA TCG CTG GAT TAC TTA TTG CTG GCT TTT TCA AGT

V L L L S L I G A S L D Y L L L A F S S 61 GCG CTT TGG ATG CTG TAT TTA GGC CGT TTG CTT TCA GGG ATC ACA GGA GCT ACT GGG GCT

A L W M L Y L G R L L S G I T G A T G A 121 GTC GCG GCA TCG GTC ATT GCC GAT ACC ACC TCA GCT TCT CAA CGC GTG AAG TGG TTC GGT

V A A S V I A D T T S A S Q R V K W F G 181 TGG TTA GGG GCA AGT TTT GGG CTT GGT TTA ATA GCG GGG CCT ATT ATT GGT GGT TTT GCA

W L G A S F G L G L I A G P I I G G F A 241 GGA GAG ATT TCA CCG CAT AGT CCC TTT TTT ATC GCT GCG TTG CTA AAT ATT GTC GCT TTC

G E I S P H S P F F I A A L L N I V A F 301 CTT GTG GTT ATG TTT TGG TTC CGT GAA ACC AAA AAT ACA CGT GAT AAT ACA GAT ACC GAA

L V V M F W F R E T K N T R D N T D T E 361 GTA GGG GTT GAG ACG CAA TCG AAT TCG GTA TAC ATC ACT TTA TTT AAA ACG ATG CCC ATT

V G V E T Q S N S V Y I T L F K T M P I 421 TTG TTG ATT ATT TAT TTT TCA GCG CAA TTG ATA GGC CAA ATT CCC GCA ACG GTG TGG GTG

L L I I Y F S A Q L I G Q I P A T V W V 481 CTA TTT ACC GAA AAT CGT TTT GGA TGG AAT AGC ATG ATG GTT GGC TTT TCA TTA GCG GGT

L F T E N R F G W N S M M V G F S L A G 541 CTT GGT CTT TTA CAC TCA G

L G L L H S

Fig. 12. Nucleotide sequence of tetracycline resistance class B (tetB) gene of tetracycline

resistant S. Weltevreden strain SW9

1 TCC CAA TGG CAT CGT AAA GAA CAT TTT GAG GCA TTT CAG TCA GTT GCT CAA TGT ACC T

S Q W H R K E H F E A F Q S V A Q C T 61 AT AAC CAG ACC GTT CAG CTG GAT ATT ACG GCC TTT TTA AAG ACC GTA AAG AAA AAT AAG

Y N Q T V Q L D I T A F L K T V K K N K 120 CAC AAG TTT TAT CCG GCC TTT ATT CAC ATT CTT GCC CGC CTG ATG AAT GCT CAT CCG GAA

H K F Y P A F I H I L A R L M N A H P E 180 TTT CGT ATG GCA ATG AAA GAC GGT GAG CTG GTG ATA TGG GAT AGT GTT CAC CCT TGT TA

F R M A M K D G E L V I W D S V H P C Y 239 C ACC GTT TTC CAT GAG CAA ACT GAA ACG TTT TCA TCG CTC TGG AGT GAA TAC CAC GAC G

T V F H E Q T E T F S S L W S E Y H D 298 AT TTC CGG CAG TTT CTA CAC ATA TAT TCG CAA GAT GTG GCG TGT TAC GGT GAA AAC CTG G

D F R Q F L H I Y S Q D V A C Y G E N L 358 CC TAT TTC CCT AAA GGG TTT ATT GAG AAT ATG TTT TTC GTC TCA GCC AAT CCC TGG GTG AG

A Y F P K G F I E N M F F V S A N P W V S 419 T TTC ACC AGT TTT GAT TTA AAC GTG GCC AAT ATG GAC AAC TTC TTC GCC CCC GTT TTC ACC

F T S F D L N V A N M D N F F A P V F T 480 ATG GGC AAA TAT TAT ACG CAA GGC GAC AAG

M G K Y Y T Q G D K

Fig. 13. Nucleotide sequence of chloramphenicol acetyltransferase (catA1) gene of

chloramphenicol resistant S. Weltevreden strain SW9

Results

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Table 13. Difference in genotypic and phenotypic resistance characteristics in Salmonella

serovars

Serovars/no. No. of isolates positive for

catA1 by PCR

No. of isolates positive for catA1 by

PCR but phenotypically negative

S. Weltevreden (17) 13 3

S. Paratyphi C (8) 4 1

S. Anatum (2) 1 -

S. Oslo (8) 4 1

S. Typhimurium (3) 1 1

S. Newport (2) - -

1 TCC CAA TGG CAT CGT AAA GAA CAT TTT GAG GCA TTT CAG TCA GTT GCT CAA TGT ACC T

S Q W H R K E H F E A F Q S V A Q C T

61 AT AAC CAG ACC GTT CAG CTG GAT ATT ACG GCC TTT TTA AAG ACC GTA AAG AAA AAT AAG

Y N Q T V Q L D I T A F L K T V K K N K 120 CAC AAG TTT TAT CCG GCC TTT ATT CAC ATT CTT GCC CGC CTG ATG AAT GCT CAT CCG GAA

H K F Y P A F I H I L A R L M N A H P E

180 TTT CGT ATG GCA ATG AAA GAC GGT GAG CTG GTG ATA TGG GAT AGT GTT CAC CCT TGT TA

F R M A M K D G E L V I W D S V H P C Y 239 C ACC GTT TTC CAT GAG CAA ACT GAA ACG TTT TCA TCG CTC TGG AGT GAA TAC CAC GAC G

T V F H E Q T E T F S S L W S E Y H D 298 AT TTC CGG CAG TTT CTA CAC ATA TAT TCG CAA GAT GTG GCG TGT TAC GGT GAA AAC CTG G

D F R Q F L H I Y S Q D V A C Y G E N L 358 CC TAT TTC CCT AAA GGG TTT ATT GAG AAT ATG TTT TTC GTC TCA GCC AAT CCC TGG GTG AG

A Y F P K G F I E N M F F V S A N P W V S 419 T TTC ACC AGT TTT GAT TTA AAC GTG GCC AAT ATG GAC AAC

F T S F D L N V A N M D N

Fig. 14. Nucleotide sequence of chloramphenicol acetyltransferase (catA1) gene of

chloramphenicol sensitive S. Weltevreden strain SW30

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Fig. 15. Detection of promoter region of catA1 gene of Salmonella serovars. Lane M : 100

bp molecular weight marker (GeneiTM

, Merck Bioscience, Bangalore); Lane 1: 528 bp

product of SW9; Lane 2: No amplification in SW30

Results

1

Fig. 16. Integron detection by PCR of Salmonella serovars DNA using: (A) Primers int1F

and int1B. Lane 1: 100 bp molecular weight marker (GeneiTM

, Merck Bioscience,

Bangalore); Lane 2: Negative control, Lane 3: 800 bp product of SW9; Lanes 4 and 5:

1200 bp product of SN36 and SN33 respectively; Lane 6: sample negative for int1F and

int1B; Lane 7: 1 kb molecular weight marker. (B) Primers qacE∆1 and sulB. Lane 1: 100

bp molecular weight marker (GeneiTM

, Merck Bioscience, Bangalore); Lane 2: Negative

control; Lanes 3, 4 and 5: 800 bp product of SW9, SN36 and SN33 respectively; Lane 6:

sample negative for qacE∆1 and sulB. (C) Primers int1F and sulB. Lane 1: 1 kb

molecular weight marker (GeneiTM

, Merck Bioscience, Bangalore); Lane 2: Negative

control; Lane 3: 1597 bp product of SW9; Lanes 4 and 5: 2200 bp product of SN36 and

SN33 respectively; Lane 6: sample negative for int1F and sulB

A B C

Results

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1 AC GAC GGG GGT CGT TTT TGG ATG TTA TGG AGC AGC AAC GAT GTT ACG CAG CAG GGC AGT C

M L W S S N D V T Q Q G S 61 GC CCT AAA ACA AAG TTA GCC ATT ACG GGG GTT GAA TTG AAA ATT TCA TTG ATT TCT GCA A

R P K T K L A I T G V E L K I S L I S A 121 CG TCA GAA AAT GGC GTA ATC GGT AAT GGC CCT GAT ATC CCA TGG TCA GCA AAA GGT GAG C

T S E N G V I G N G P D I P W S A K G E 181 AG TTA CTC TTT AAA GCG CTC ACA TAT AAT CAG TGG CTC CTT GTT GGA AGG AAA ACA TTT G

Q L L F K A L T Y N Q W L L V G R K T F 241 AC TCT ATG GGT GTT CTT CCA AAT CGA AAA TAT GCA GTA GTG TCG AGG AAA GGA ATT TCA A

D S M G V L P N R K Y A V V S R K G I S 301 GC TCA AAT GAA AAT GTA TTA GTC TTT CCT TCA ATA GAA ATC GCT TTG CAA GAA CTA TCG A

S S N E N V L V F P S I E I A L Q E L S 361 AA ATT ACA GAT CAT TTA TAT GTC TCT GGT GGC GGT CAA ATC TAC AAT AGT CTT ATT GAA A

K I T D H L Y V S G G G Q I Y N S L I E 421 AA GCA GAT ATA ATT CAT TTG TCT ACT GTT CAC GTT GAG GTT GAA GGT GAT ATC AAT TTT C

K A D I I H L S T V H V E V E G D I N F 481 CT AAA ATT CCA GAG AAT TTC AAT TTG GTT TTT GAG CAG TTT TTT TTG TCT AAT ATA AAT T

P K I P E N F N L V F E Q F F L S N I N 541 AC ACA TAT CAG ATT TGG AAA AAA GGC TAA CAA GTC GTT CCA GCA CCA GTC GCT GCG CTC C

Y T Y Q I W K K G * 601 TT GGA CAG TTT TTA AGT CGC GGT TTT ATG GTT TTG CTG CGC AAA AGT ATT CCA TAA AAC C

661 AC AAC TTA AAA ACT GCC GCT GAA CTC GGC GTT AGA TGC ACT AAG CAC ATA ATT GCT CAC A

721 GC CAA ACT ATC AGG TCA AGT CTG CTT TTA TTA TTT TTA AGC GTG CAT AAT AAG CCC TAC A

781 CAA ATT GGG AGA TAT ATC ATG AAA GGC TGG CTT TTT CTT GTT ATC GCA ATA GTT GGC GAA

M K G W L F L V I A I V G E

841 GTA ATC GCA ACA TCC GCA TTA AAA TCT AGC GAG GGC TTT ACT AAG CTT GCC CCT TCC GCC

V I A T S A L K S S E G F T K L A P S A 901 GTT GTC ATA ATC GGT TAT GGC ATC GCA TTT TAT TTT CTT TCT CTG GTT CTG AAA TCC ATC

V V I I G Y G I A F Y F L S L V L K S I

961 CCT GTC GGT GTT GCT TAT GCA GTC TGG TCG GGA CTC GGC GTC GTC ATA ATT ACA GCC ATT

P V G V A Y A V W S G L G V V I I T A I 1021 GCC TGG TTG CTT CAT GGG CAA AAG CTT GAT GCG TGG GGC TTT GTA GGT ATG GGG CTC ATA

A W L L H G Q K L D A W G F V G M G L I

1081 ATT GCT GCC TTT TTG CTC GCC CGA TCC CCA TCG TGG AAG TCG CTG CGG AGG CCG ACG CCA

I A A F L L A R S P S W K S L R R P T P 1141 TGGTGACG GTG TTC GGC ATT CTG AAT CTC ACC GAG GAC TCC TTC TTC GAT GAG AGC CGG C

W *

M V T V F G I L N L T E D S F F D E S R 1201 GG CTA GAC CCC GCC GGC GCT GTC ACC GCG GCG ATC GAA ATG CTG CGA GTC GGA TCA GAC G

R L D P A G A V T A A I E M L R V G S D 1261 TC GTG GAT GTC GGA CCG GCC GCC AGC CAT CCG GAC GCG AGG CCT GTA TCG CCG GCC GAT G

V V D V G P A A S H P D A R P V S P A D 1321 AG ATC AGA CGT ATT GCG CCG CTC TTA GAC GCC CTG TCC GAT CAG ATG CAC CGT GTT TCA A

E I R R I A P L L D A L S D Q M H R V S 1381 TC GAC AGC TTC CAA CCG GAA ACC CAG CGC TAT GCG CTC AAG CGC GGC GTG GGC TAC CTG A

I D S F Q P E T Q R Y A L K R G V G Y L 1441 AC GAT ATC CAA GGA TTT CCT GAC CCT GCG CTC TAT CCC GAT ATT GCT GAG GCG GAC TGC A

N D I Q G F P D P A L Y P D I A E A D C 1501 GG CTG GTG GTT ATG CAC TCA GCG CAG CGG GAT GGC ATC GCC ACC CGC ACC GGT CAC CTT C

R L V V M H S A Q R D G I A T R T G H L 1561 GA CCC GAA GAC GCG TTC GAC CCG AAA AAG AAG TAC CC

R P E D A F D P K K K Y P

Results

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Fig. 17. Nucleotide sequence of S. Weltevreden strain SW9 class I integron dihydrofolate

reductase type A7 (dhfrA7), Quaternary ammonium compound and disinfectant

resistance protein (qacEdelta1), sulphonamide resistance protein (sul1delta). The

translated amino acid sequences of the individual integron gene cassettes are shown

under the nucleotide sequences. The putative transcription start codon and stop codons

are underlined

Results

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1 GTT TGA TGT TTG GAG CAG CAA CGA TGT TAC GCA GCA GGG CAG TCG CCC TAA AAC AAA GTT

61 AAC CTC TGA GGA AGA ATT GTG AAA CTA TCA CTA ATG GTA GCT ATA TCG AAG AAT GGA GTT

V K L S L M V A I S K N G V 121 ATC GGG AAT GGC CCT GAT ATT CCA TGG AGT GCC AAA GGT GAA CAG CTC CTG TTT AAA GCT

I G N G P D I P W S A K G E Q L L F K A 181 ATT ACC TAT AAC CAA TGG CTG TTG GTT GGA CGC AAG ACT TTT GAA TCA ATG GGA GCA TTA

I T Y N Q W L L V G R K T F E S M G A L 241 CCC AAC CGA AAG TAT GCG GTC GTA ACA CGT TCA AGT TTT ACA TCT GAC AAT GAG AAC GTA

P N R K Y A V V T R S S F T S D N E N V 301 TTG ATC TTT CCA TCA ATT AAA GAT GCT TTA ACC AAC CTA AAG AAA ATA ACG GAT CAT GTC

L I F P S I K D A L T N L K K I T D H V 361 ATT GTT TCA GGT GGT GGG GAG ATA TAC AAA AGC CTG ATC GAT CAA GTA GAT ACA CTA CAT

I V S G G G E I Y K S L I D Q V D T L H 421 ATA TCT ACA ATA GAC ATC GAG CCG GAA GGT GAT GTT TAC TTT CCT GAA ATC CCC AGC AAT

I S T I D I E P E G D V Y F P E I P S N 481 TTT AGG CCA GTT TTT ACC CAA GAC TTC GCC TCT AAC ATA AAT TAT AGT TAC CAA ATC TGG

F R P V F T Q D F A S N I N Y S Y Q I W 541 CAA AAG GGT TAA CAA GTG GCA GCA ACG GAT TCG CAA ACC TGT CAC GCC TTT TGT ACC AAA

Q K G * 601 AGC CGC GCC AGG TTT GCG ATC CGC TGT GCC AGG CGT TAA GGC TAC ATG AAA ATC GTA CAT

M K I V H 661 TAC GAA GCG AAT GCA CCA TGG ATA GGA AGA ATG AAA TGC CCA AAC CCA AAG TGT GGG AAG

Y E A N A P W I G R M K C P N P K C G K

721 GAA ACT CCT GCC TGG CAA TCG AGC GGC ATG AGC GAC AGT TGC CCG CAT TTT TTC TGT GAT

E T P A W Q S S G M S D S C P H F F C D 781 ACT TGC TCG AAT GTA ATC CAT AGA GAG CAG GAC CAT GCA TTA CTG TAT GAA AAT GAA ATC

T C S N V I H R E Q D H A L L Y E N E I

841 AAT CAA GAG CTC TTG GAT CGA ATA GCA GCA ACT CTT CCA GAT TGC CCT TGC GGG GGT AGG

N Q E L L D R I A A T L P D C P C G G R 901 TTT GTT CCT GGT GCA AAC CCA AAG TGT CCG AGT TGC AAG ACC GAG TAC GTG CAC CAA TGG

F V P G A N P K C P S C K T E Y V H Q W

961 GAT GCA GTG AAA AGG TTG AAT GTA CCT TTT ATG CCA ATC TTG GAT GGT TCC TGC TTG ATT

D A V K R L N V P F M P I L D G S C L I 1021 CGA GAT AGG CTG TAT TCG TAT GAA GTA TGC ATT GGT TCT AAA CCA AAA TAC TGG TGG CGT

R D R L Y S Y E V C I G S K P K Y W W R

1081 TTG TTC ACA AAT GCC TTA ACA AGT TTA GGC AAG GGA CGC TCC TGA GTC GCG CCC CTG CT

L F T N A L T S L G K G R S * 1141 A AAA GCG TTA GAT GCA CTA AGC ACA TAA TTG CTC ACA GCC AAA CTA TCA GGT CAA GTC TG

1201 C TTT TAT TAT TTT TAA GCG TGC ATA ATA AGC CCT ACA CAA ATT GGG AGA TAT ATC ATG AA

M K 1261 A GGC TGG CTT TTT CTT GTT ATC GCA ATA GTT GGC GAA GTA ATC GCA ACA TCC GCA TTA AA

G W L F L V I A I V G E V I A T S A L K

1321 A TCT AGC GAG GGC TTT ACT AAG CTT GCC CCT TCC GCC GTT GTC ATA ATC GGT TAT GGC AT

S S E G F T K L A P S A V V I I G Y G I 1381 C GCA TTT TAT TTT CTT TCT CTG GTT CTG AAA TCC ATC CCT GTC GGT GTT GCT TAT GCA GT

A F Y F L S L V L K S I P V G V A Y A V

1441 C TGG TCG GGA CTC GGC GTC GTC ATA ATT ACA GCC ATT GCC TGG TTG CTT CAT GGG CAA AA

W S G L G V V I I T A I A W L L H G Q K 1501 G CTT GAT GCG TGG GGC TTT GTA GGT ATG GGG CTC ATA ATT GCT GCC TTT TTG CTC GCC CG

L D A W G F V G M G L I I A A F L L A R

1561 A TCC CCA TCG TGG AAG TCG CTG CGG AGG CCG ACG CCATGGTGACG GTG TTC GGC ATT CTG

S P S W K S L R R P T P W *

M V T V F G I L 1621 AAT CTC ACC GAG GAC TCC TTC TTC GAT GAG AGC CGG CGG CTA GAC CCC GCC GGC GCT GTC

N L T E D S F F D E S R R L D P A G A V

1681 ACC GCG GCG ATC GAA ATG CTG CGA GTC GGA TCA GAC GTC GTG GAT GTC GGA CCG GCC GCC

Results

Page 105

T A A I E M L R V G S D V V D V G P A A 1741 AGC CAT CCG GAC GCG AGG CCT GTA TCG CCG GCC GAT GAG ATC AGA CGT ATT GCG CCG CTC

S H P D A R P V S P A D E I R R I A P L

1801 TTA GAC GCC CTG TCC GAT CAG ATG CAC CGT GTT TCA ATC GAC AGC TTC CAA CCG GAA ACC

L D A L S D Q M H R V S I D S F Q P E T 1861 CAG CGC TAT GCG CTC AAG CGC GGC GTG GGC TAC CTG AAC GAT ATC CAA GGA TTT CCT GAC

Q R Y A L K R G V G Y L N D I Q G F P D

1921 CCT GCG CTC TAT CCC GAT ATT GCT GAG GCG GAC TGC AGG CTG GTG GTT ATG CAC TCA GCG

P A L Y P D I A E A D C R L V V M H S A 1981 CAG CGG GAT GGC ATC GCC ACC CGC ACC GGT CAC CTT CGA CCC GAA GAC GCT TCT CGA TTA

Q R D G I A T R T G H L R P E D A S R L

2041 GAG AAT GCG GTT CTG

E N A V L

Fig. 18. Nucleotide sequence of S. Newport strain SN36 / SN33 class I integron

dihydrofolate reductase type A1 (dhfrA1), OrfC, Quaternary ammonium compound and

disinfectant resistance protein (qacEdelta1)/Orf3, sulphonamide resistance protein

(sul1delta). The translated amino acid sequences of the individual integron gene cassettes

are shown under the nucleotide sequences. The putative transcription start codon and

stop codons are underlined

Results

Page 106

Fig. 19. (A) Map of integron of S. Weltevreden (B) Map of integron of S. Newport to

show the approximate position of primer (Table 4) sites and regions of amplified DNA.

The 5'CS (containing the intI1 gene) and the 3'CS (containing the intI1 gene) are shown

by boxes. The individual gene cassettes are shown together with their recombination

sites, with a 59 bp element indicated by an ellipse. The dfrA1/orfC and dfrA7 genes confer

resistance to co-trimoxazole (trimethoprim + sulphamethoxazole), qacEΔ1 confer

resistance to quaternary ammonium compounds and sul1 confer resistance to

sulphonamide are shown in arrows

A

B

Results

Page 107

4.2. Multiplex PCR assay for the detection of antibiotic resistance as well as virulence

genes of Salmonella pathogenicity island 2

4.2.1. Multiplex PCR

In this study, a specific mPCR to detect multidrug resistance Salmonella from seafood

in a single step has been developed and evaluated. All Salmonella strains listed in Table 14

were tested for drug resistance using disc diffusion method. Three isolates of S. Newport and

one of S. Weltevreden were resistant to sulphonamide. Twelve isolates of S. Weltevreden and

two S. Typhimurium were resistant to florfenicol. One isolate of S. Newport which was

resistant to sulphonamide was also resistant to tetracycline and florfenicol thus conferring the

isolate as multidrug resistant (Table 15). Remaining isolates though negative for antibiotic

resistance genes, were positive for SPI-2 genes as well as for invasion gene by mPCR.

4.2.2. Uniplex PCR for Salmonella

Uniplex PCR was performed using six sets of primers targeting one genus specific

gene (invA), two SPI-2 genes (ssaT and sseF) and three multidrug resistance genes (sul1, tetG

and floR) of Salmonella. All products generated were expected of the amplicon size when

tested with the representative isolate of S. Newport (Fig. 20). PCR amplification of the six sets

of genes by uniplex PCR gave reproducible results.

Results

Page 108

Table 14. Bacterial strains in the present study and results by mPCR assay

Bacterial strains

No.of isolates

mPCR

invA Sul1 tetG ssaT sseF floR

S. Typhimurium 10 10 - - 10 10 2

S. Weltevreden 19 19 1 - 19 19 12

S. Oslo 11 11 - - 11 11 -

S. Newport 10 10 3 1 10 10 1

S. Bareilly 6 6 - - 6 6 -

S. Stanley 1 1 - - 1 1 -

S. Infantis 3 3 - - 3 3 -

S. Virchow 1 1 - - 1 1 -

S. Paratyphi 4 4 - - 4 4 -

Non-Salmonella strains

Escherichia coli 3 - - - - - -

Vibrio parahaemolyticus 5 - - - - - -

V. harveyi 4 - - - - - -

V. alginolyticus 1 - - - - - -

V. anguillarum 1 - - - - - -

V. campbellii 1 - - - - - -

Staphylococcus epedermidis 1 - - - - - -

S. aureus 1 - - - - - -

Bacillus cereus 1 - - - - - -

B. coagulans 1 - - - - - -

Aeromonas hyrdrophila 3 - - - - - -

Results

Page 109

Table 15. Detection of various antimicrobial resistance types by disk diffusion and

mPCR method

Type of resistance Number of isolates detected by

Disk diffusion mPCR

TSuF 1 1

SuF 12 15

T: Tetracycline, Su: Sulphonamide, F: Florfenicol

Fig. 20. Amplification profiles of the primer sets for the mPCR assay. S. Newport

template DNA was used in the seven lanes of the gel, with the primer pairs indicated

above each lane. 100 bp DNA ladder (GeneiTM

, Merck Bioscience, Bangalore) was used

in the lane M. Amplicon sizes for primer pairs are given in base pairs to the left of the gel

Results

Page 110

4.2.3. Optimization of mPCR assay

Amplification of all six sets of primers simultaneously was achieved with comparable

band intensities at annealing temperature of 55 °C (Table 6). Gradient PCR helped in best

annealing temperature to amplify the DNA products for developing mPCR. An annealing

temperature of 55 °C was taken as an optimum temperature for the PCR amplification.

4.2.4. Sensitivity of mPCR

In this study comparison of different homogenates comprising of fish, shrimp and clam

was done for sensitivity test. Salmonella was detected after 4 h in shrimp homogenates

inoculated with 10-1

(9.45×107

CFU/ml) and 10-2

(9.45×106

CFU/ml) dilutions of Salmonella

(Table 16). But, 6 h and 8 h pre-enrichment resulted in detectable amplicons at even lower

inoculum levels of 10-3

(9.45×105

CFU/ml) and 10-4

(9.45×104

CFU/ml) dilutions respectively.

The 10-6

dilution corresponds to 9.45×102

CFU/ml was detectable only after 20 h of

incubation. In case of fish homogenates, Salmonella was detected after 8 h incubation with 10-

1 (9.83×10

7CFU/ml) to 10

-4 (9.83×10

4CFU/ml) dilutions. But, 20 h pre-enrichment resulted in

detectable amplicons at even lower inoculum levels of 10-8

(9.83 CFU/ml) dilutions. Whereas,

in case of clam homogenates Salmonella was detected immediately after incubation at 0 h

with 10-1

(9.79×107

CFU/ml) to 10-2

(9.79×106

CFU/ml) dilutions. But, 20 h pre-enrichment

resulted in detectable amplicons at even lower inoculum levels of 10-8

(9.79 CFU/ml)

dilutions. The results were compared with culture grown in lactose broth that gave exact

amplification even at lower inoculum level of 10-3

(9.1×105

CFU/ml) dilutions at 0 h

incubation time and 10-8

(9.1 CFU/ml) dilutions at 4 h incubation time. The results also show

that the type of sample (fish, shrimp and clam) would also play an important role in detection

Results

Page 111

efficiency of multidrug resistant Salmonella. The reason for difference in the detection level of

Salmonella spp. in different samples need further study. The method developed proved to b e

sensitive and rapid. The degree of specificity and sensitivity of the mPCR assay of spiked

seafood sample was high, and the assay was able to detect Salmonella from fish and clam

homogenates even with as few as ~9 CFU/ml when compared to shrimp homogenates with

~9.45×102

CFU/ml cells. When tested on tenfold serially diluted bacterial genomic DNA, the

limit of detection of the mPCR assay for S. enterica serovar Newport was 1 ng/µL of original

dilution (Fig. 21).

4.2.5. Specificity of mPCR assay

This report describes a sensitive, rapid, and validated mPCR assay for the detection of

Salmonella from seafood. The three sets of primers (invA, ssaT, sseF) employed in the study

are highly specific for Salmonella. This indicates that each of the selected oligonucleotide

primers for each of the targeted gene segments was specific for the genus Salmonella. The

specificity of these oligonucleotides was further affirmed by PCR amplification of 65

serologically confirmed Salmonella isolates from seafood and clinical samples and 22 non

Salmonella cultures (Table 14). No amplification was resulted even with the antibiotic

resistance primers from non Salmonella isolates. The optimum annealing temperature of 55 °C

proved to be adequate for mPCR reaction preventing nonspecific reactions.

Results

Page 112

Table 16. Detection of S. Newport in artificially contaminated fish, shrimp and mollusc

samples by mPCR assay

Sample type

Duration of

pre-enrichment (h)

Bacterial dilution

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

Shrimp

(9.45×108

CFU/ml)

0 - - - - - - - -

2 - - - - - - - -

4 + + - - - - - -

6 + + + - - - - -

8 + + + + - - - -

20 + + + + + + - -

Fish

(9.83×108

CFU/ml)

0 - - - - - - - -

2 - - - - - - - -

4 - - - - - - - -

6 - - - - - - - -

8 + + + + - - - -

20 + + + + + + + +

Mollusc

(9.79×108

CFU/ml)

0 + + - - - - - -

2 + + - - - - - -

4 + + + - - - - -

6 + + + + - - - -

8 + + + + + - - -

20 + + + + + + + +

Lactose

Broth

(9.1×108

CFU/ml)

0 + + + - - - - -

2 + + + + - - - -

4 + + + + + + + +

6 + + + + + + + +

8 + + + + + + + +

20 + + + + + + + +

Results

Page 113

Fig. 21. Sensitivity of mPCR assay on tenfold serially diluted S. Newport DNA. Lane M:

100 bp DNA ladder (GeneiTM

, Merck Bioscience, Bangalore), Lane P: Positive control;

Lanes 1-5 represent different concentrations of serovar Newport DNA from 1000 ng/µl

to 0.1 ng/µl respectively; Lane N: Negative control

Results

Page 114

4.3. Investigation of difference in expression of invasion genes of SPI-1, growth, infection,

replication and cytotoxicity of nalidixic acid (quinolone) resistant isolates from those of

susceptible isolates

4.3.1. Bacteria susceptibility to nalidixic acid

Out of two susceptible and two resistance isolates of Salmonella, SN71 (Fig. 22A) and

SW30 (Fig. 23A) were susceptible to nalidixic acid with a MIC of < 4µg/ml and SN36 (Fig.

22B) and SW9 (Fig. 23B) were resistance to nalidixic acid with MIC of > 4µg/ml. Two

isolates of S. Newport (SN71, SN36) and two S. Weltevreden (SW30, SW9) were selected for

the further study.

4.3.2. Selection of nalidixic acid resistant mutants

One isolate of each S. Weltevreden 30 (SW30 with MIC < 4µg/ml) and S. Newport 71

(SN71 with MIC < 4µg/ml) were used as the parental strains. S. Newport (SN71) and S.

Weltevreden (SW30) which were previously sensitive to nalidixic acid, was made resistant by

multiple passage selection through increasing concentrations of nalidixic acid in vitro and

given a code of SN71R (Fig. 22C) and SW30R (Fig. 23C) (MIC >256 µg/ml).

Results

Page 115

Fig. 22. (A) Represents nalidixic acid sensitive S. Newport (SN71) with an MIC of 3

µg/ml using Ezy MICTM

strips, (B) Represents wild nalidixic acid resistant S. Newport

(SN36) showing no MIC for nalidixic acid, (C) Represents nalidixic acid resistant S.

Newport (SN71R) after inducing resistance

A B

C

Results

Page 116

Fig. 23. (A) Represents nalidixic acid sensitive S. Weltevrden (SW30) with an MIC of 3

µg/ml using Ezy MICTM

strips, (B) Represents wild nalidixic acid resistant S. Weltevrden

(SW9) showing no MIC for nalidixic acid, (C) Represents nalidixic acid resistant S.

Weltevrden (SW30R) after inducing resistance

A B

C

Results

Page 117

4.3.3. Bacterial growth

Growth kinetics of nalidixic acid resistance isolates of Salmonella (SN36, SW9,

SN71R and SW30R) was significantly (< 0.001) slower than the nalidixic acid sensitive

strains (SN71 and SW30) (Fig. 24 and Fig. 25). The multiplication rate of nalidixic acid

resistance isolates was lesser than that of susceptible isolates under the same culture

conditions. The culture density of nalidixic acid resistance strains was always lower than that

of susceptible strains.

4.3.4. Analysis of quinolone resistance determining region (QRDR) from SN36, SN71,

SN71R, SW9, SW30 and SW30R

All the tested isolates harbored gyrA, gyrB, parC and parE genes (Figs. 26A, B and C).

Plasmid mediated quinolone resistance genes were not observed in any of the strains although

plasmid mediated quinolone efflux pump was present in all isolates. A single gyrA mutation at

codon 83 (Ser to Tyr) was observed in SW9 (Fig. 27) whereas single mutation at codon 87

(Asp to Asn) was observed in SN36 (Fig. 28) resistant strains. No point mutation was

observed in SW30 and SN71 (Fig. 29) gyrA gene. After inducing resistance to SW30 and

SN71, the gyrA gene showed point mutation at codon 87 (Asp to Gly) in SN71R (Fig. 30) and

no mutation was observed in gyrA of SW30R (Fig. 31) (Table 17). There was no mutations

even in the gyrB, parC and parE of the QRDR region of any of tested isolates.

The gyrA sequence of 2 resistant (SW9 and SN36), 2 susceptible (SW30 and SN71)

and 2 isolates (SW30R and SN71R) after inducing resistance has been submitted to the NCBI

GenBank and has been assigned GenBank accession numbers (SN36- JX197394, SW9-

JX197395, SN71- JX197398, SW30, SW30R- JX197397 and SN71R- KC121321). The

Results

Page 118

sequences of gyrB, parC and parE of SW30R (Figs. 32, 33 and 34) and SW9 (Figs. 35, 36 and

37) was also submitted to the NCBI GenBank and has been assigned GenBank accession

numbers (KC121322, KC121323 and KC121324 for gyrB, parC and parE of SW30R and

KC121325, KC121326 and KC121327 for gyrB, parC and parE of SW9).

In order to identify the reason for resistance mechanism in SW30R (after inducing

resistance), susceptibility to nalidixic acid in the presence and absence of 80 µg/ml of the

efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) was examined. It was found that

growth rate of SW30R with nalidixic acid and PAβN has been reduced drastically when

compared to SW30R with nalidixic acid alone. SW30R (which was completely resistant to

nalidixic acid after inducing resistance) showed a MIC of 48µg/ml for nalidixic acid in the

presence of efflux pump inhibitor PAβN and a MIC of 24µg/ml was observed for nalidixic

acid for SN71R in the presence of PAβN.

4.3.5. Cell Envelope Protein analysis by SDS-PAGE

A cell envelope protein extract was obtained from all the tested strains (SN36, SN71,

SN71R, SW9, SW30 and SW30R) and the sample of each was run in a SDS-PAGE (Fig. 38).

The resulting gel confirmed the similar expression of cell envelope proteins in sensitive

(SN71 and SW30) as well as resistant (SN71R and SW30R) strains.

4.3.6. Epithelial cell invasiveness and intracellular replication

The epithelial cell invasion and intracellular replication in epithelial cells was

markedly reduced in wild quinolone resistant strains (SN36, SW9) as well as after inducing

resistance (SN71R, SW30R) when compared to the quinolone susceptible strains (SN71,

Results

Page 119

SW30) (Figs. 39, 40, 41 and 42). In the epithelial cell invasion assays, the number of

intracellular bacteria at 2 h and the ratio of bacteria number at 16 h relative to that at 2 h for

quinolone resistant strains were 2 fold lower, respectively, compared to wild type, quinolone

susceptible strains.

Results

Page 120

Fig. 24. Growth curve of S. Newport resistant (SN36), sensitive (SN71) and after

inducing resistance (SN71R) strain

Fig. 25. Growth curve of S. Weltevreden resistant (SW9), sensitive strain (SW30) and

after inducing resistance (SW30R) strain

Results

Page 121

Fig. 26. Quinolne resistance determining regions (QRDR) of Salmonella: (A) Lane M:

100 bp molecular weight marker (GeneiTM

, Merck Bioscience, Bangalore); Lane 1-5:

gyrA region of Salmonella; Lane 6: Negative control. (B) Lane M: 100 bp molecular

weight marker (GeneiTM

, Merck Bioscience, Bangalore) ; Lane 1-3: gyrB region of

salmonella; Lanes 4-6: parC region of Salmonella; Lane 7: Negative control (C) Lane M:

100 bp molecular weight marker (GeneiTM

, Merck Bioscience, Bangalore); Lane 1-3:

parE region of Salmonella; Lane 4: Negative control

A

B

C

Results

Page 122

1 CTG AAG CCG GTA CAC CGT CGC GTA CTT TAC GCC ATG AAC GTA TTG GGC AAT GAC TGG

L K P V H R R V L Y A M N V L G N D W

58 AAC AAA GCC TAT AAA AAA TCT GCC CGT GTC GTT GGT GAC GTA ATC GGT AAA TAC CAT

N K A Y K K S A R V V G D V I G K Y H

115 CCC CAC GGC GAT TAC GCA GTG TAT GAC ACC ATC GTT CGT ATG GCG CAG CCA TTC TCG

P H G D Y A V Y D T I V R M A Q P F S

172 CTG CGT TAC ATG CTG GTG GAT GGT CAG GGT AAC TTC GGT TCT ATT GAC GGC GAC TCC

L R Y M L V D G Q G N F G S I D G D S

229 GCG GCG GCA ATG CGT TAT ACG GAG ATC CGT CTG GCG AAA ATC GCC CAC GAA CTG AT

A A A M R Y T E I R L A K I A H E L M

285 G GCC GA

A

Fig. 27. Nucleotide sequence of nalidixic acid resistant S. Weltevreden strain SW9

quinolone resistance determining region (QRDR), with single point mutation in the gyrA

gene altering amino acid sequence from serine (S) to tyrosine (Y) (i.e TCC to TAC). The

translated amino acid sequences of the gene are shown under the nucleotide sequences





115 CCC CAC GGC GAT TCC GCA GTG TAT AAC ACC ATC GTT CGT ATG GCG CAG CCA TTC TCG

P H G D S A V Y N T I V R M A Q P F S





285 G GCC GA

A

Fig. 28. Nucleotide sequence of nalidixic acid resistant S. Newport strain SN36/SN35

quinolone resistance determining region (QRDR), with single point mutation in the gyrA

gene altering the amino acid sequence from aspartic acid (D) to aspargine (N) (i.e GAC

to AAC). The translated amino acid sequences of the gene are shown under the

nucleotide sequences

Results

Page 123





115 CCC CAC GGC GAT TCC GCA GTG TAT GAC ACC ATC GTT CGT ATG GCG CAG CCA TTC TCG

P H G D S A V Y D T I V R M A Q P F S





285 G GCC GA

A

Fig. 29. Nucleotide sequence of nalidixic acid sensitive S. Weltevreden (SW30) and S.

Newport (SN71) quinolone resistance determining region (QRDR), without any point

mutation in gyrA gene. The translated amino acid sequences of the gene are shown under

the nucleotide sequences





115 CCC CAC GGC GAT TCC GCA GTG TAT GGC ACC ATC GTT CGT ATG GCG CAG CCA TTC TCG

P H G D S A V Y G T I V R M A Q P F S





285 G GCC GA

A

Fig. 30. Nucleotide sequence of S. Newport strain after inducing nalidixic acid resistance

(SN71R), containing quinolone resistance determining region (QRDR) with a single point

mutation in gyrA gene altering amino acid sequence from aspartic acid (D) to Glycine

(G) (i.e GAC to GGC). The translated amino acid sequences of the gene are shown under

the nucleotide sequences

Results

Page 124





115 CCC CAC GGC GAT TCC GCA GTG TAT GAC ACC ATC GTT CGT ATG GCG CAG CCA TTC TCG

P H G D S A V Y D T I V R M A Q P F S





285 G GCC GA

A

Fig. 31. Nucleotide sequence of S. Weltevreden after inducing nalidixic acid resistance

(SW30R), containing quinolone resistance determining region (QRDR) without any

point mutation in gyrA gene. The translated amino acid sequences of the gene are shown

under the nucleotide sequences

Table 17. Point mutation in the QRDR of gyrA of nalidixic acid-resistant Salmonella

Point mutation in the QRDR of gyrA MIC (µg/ml) for nalidixic

acid

Nalidixic acid resistant S. Weltevreden (SW9)

Serine Tyrosine (codon 83) (TCC TAC)

Nalidixic acid resistant S. Newport (SN36 and SN33)

Aspartic acid Aspargine (codon 87) (GAC AAC)

Nalidixic acid resistant after induction S. Newport (SN71R)

Aspartic acid Glycine (codon 87) (GAC GGC)

Nalidixic acid resistant after induction S. Weltevreden (SW30R)

No point mutation

>256

>256

>256

>256

Results

Page 125

1 TTC CGG CAT CTG ACG ATA GAA GAA GGT CAA CAG CAG CGT ACG GAT GTG CGA GCC

F R H L T I E E G Q Q Q R T D V R A

55 GTC GAC GTC CGC ATC GGT CAT GAT GAT GAT GCT GTG ATA GCG CAG CTT GTC CGG G

V D V R I G H D D D A V I A Q L V R

110 TT GTA CTC GTC GCG ACC GAT ACC GCA GCC CAG CGC GGT GAT CAG CGT CGC CAC TT

V V L V A T D T A A Q R G D Q R R H F

165 C CTG GGA GGA AAG CAT CTT GTC GAA GCG CGC TTT CTC GAC GTT AAG GAT TTT ACC T

L G G K H L V E A R F L D V K D F T

220 TT CAG CGG CAG AAT CGC CTG GTT CTT GCG GTT ACG CCC CTG CTT CGC AGA GCC GCC

F Q R Q N R L V L A V T P L L R R A A

277 CGC GGA GTC CCC TTC CAC CAG GTA CAG TTC GGA CAG CGC CGG GTC GCG TTC CTG A

R G V P F H Q V Q F G Q R R V A F L

332 CA GTC CGC CAG TTT G

T V R Q F

Fig. 32. Nucleotide sequence of S. Weltevreden quinolone resistance determining region

(QRDR) after inducing nalidixic acid resistance (SW30R), without any point mutation in

gyrB gene. The translated amino acid sequences of the gene are shown under the


1 GCC TAC TTA AAC TAC TCC ATG TAC GTG ATC ATG GAT CGT GCG TTG CCG TTT ATT GGC GAC

A Y L N Y S M Y V I M D R A L P F I G D

61 GGC CTG AAG CCG GTA CAG CGC CGC ATC GTC TAT GCG ATG TCA GAG CTG GGG CTG AAC GC

G L K P V Q R R I V Y A M S E L G L N A

120 C ACC GCT AAA TTT AAA AAA TCC GCC CGT ACC GTT GGC GAC GTA CTG GGT AAG TAT CAC C

T A K F K K S A R T V G D V L G K Y H

179 CG CAT GGC GAC ATC GCC TGC TAT GAA GCC ATG GTG CTG ATG GCG CAG CCG TTC TCT TAC

P H G D I A C Y E A M V L M A Q P F S Y

238 CGT TAC CCG CTG GTC GAT GGC CAG GGG AAT TGG GGC GCG CCG GAT GAT CCG AAG TCA TT

R Y P L V D G Q G N W G A P D D P K S F

297 C GCG GCG ATG CGT TAT ACC GAA TCC CGC CTG TCC AAA TAC GCC GAG CTG CTG TTA AGC G

A A M R Y T E S R L S K Y A E L L L S

356 AA CTC GGT CAG GGG ACT GCG GAC TGG GTG CCA AAC TTC GAC GGC ACC ATG CAG GAA CC

E L G Q G T A D W V P N F D G T M Q E P

414 G AAA ATG TTA CCG GCG CGT CTG CCG AAC ATC CTG CTG AAC GGC ACC ACC GGT ATT GCG G

K M L P A R L P N I L L N G T T G I A

473 TG GGC ATG GCA ACA GAT ATT CCG CCG CAT AAC CTG CGC GAA GTC GCG AAA GCG GCG A

V G M A T D I P P H N L R E V A K A A

530 TT ACG CTG AT

I T L


(QRDR) after inducing nalidixic acid resistance (SW30R) without any point mutation in

topoisomerase IV (parC) gene. The translated amino acid sequences of the gene are

shown under the nucleotide sequences

Results

Page 126

1 ACC GAG CTG TTC CTT GTG GAA GGG GAT TCG GCG GGC GGT TCC GCC AAG CAG GCG CGC GAT

T E L F L V E G D S A G G S A K Q A R D 61 CGC GAA TAT CAG GCG ATC ATG CCG CTC AAA GGT AAG ATC CTT AAC ACC TGG GAG GTC TCT T

R E Y Q A I M P L K G K I L N T W E V S 122 CC GAT GAA GTG CTG GCC TCG CAA GAA GTG CAT GAT ATT TCC GTG GCG ATC GGT ATC GAT CC

S D E V L A S Q E V H D I S V A I G I D P 183 G GAC AGC GAC GAT CTG AGT CAG CTG CGC TAC GGC AAG ATC TGT ATC CTG GCG GAT GCG GA

D S D D L S Q L R Y G K I C I L A D A D 243 C TCC GAT GGT TTG CAT ATC GCT ACT CTG CT

S D G L H I A T L L


(QRDR) after inducing nalidixic acid resistance (SW30R), without any point mutation in

topoisomerase IV (parE) gene. The translated amino acid sequences of the gene are

shown under the nucleotide sequences

1 TTC CGG CAT CTG ACG ATA GAA GAA GGT CAA CAG CAG CGT ACG GAT GTG CGA GCC

F R H L T I E E G Q Q Q R T D V R A

55 GTC GAC GTC CGC ATC GGT CAT GAT GAT GAT GCT GTG ATA GCG CAG CTT GTC CGG G

V D V R I G H D D D A V I A Q L V R

110 TT GTA CTC GTC GCG ACC GAT ACC GCA GCC CAG CGC GGT GAT CAG CGT CGC CAC TT

V V L V A T D T A A Q R G D Q R R H F

165 C CTG GGA GGA AAG CAT CTT GTC GAA GCG CGC TTT CTC GAC GTT AAG GAT TTT ACC T

L G G K H L V E A R F L D V K D F T

220 TT CAG CGG CAG AAT CGC CTG GTT CTT GCG GTT ACG CCC CTG CTT CGC AGA GCC GCC

F Q R Q N R L V L A V T P L L R R A A

277 CGC GGA GTC CCC TTC CAC CAG GTA CAG TTC GGA CAG CGC CGG GTC GCG TTC CTG A

R G V P F H Q V Q F G Q R R V A F L

332 CA GTC CGC CAG TTT G

T V R Q F

Fig. 35. Nucleotide sequence of nalidixic acid resistant S. Weltevreden (SW9) quinolone

resistance determining region (QRDR), without any point mutation in gyrB gene. The

translated amino acid sequences of the gene are shown under the nucleotide sequences

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1 GCC TAC TTA AAC TAC TCC ATG TAC GTG ATC ATG GAT CGT GCG TTG CCG TTT ATT GGC GAC

A Y L N Y S M Y V I M D R A L P F I G D

61 GGC CTG AAG CCG GTA CAG CGC CGC ATC GTC TAT GCG ATG TCA GAG CTG GGG CTG AAC GC

G L K P V Q R R I V Y A M S E L G L N A

120 C ACC GCT AAA TTT AAA AAA TCC GCC CGT ACC GTT GGC GAC GTA CTG GGT AAG TAT CAC C

T A K F K K S A R T V G D V L G K Y H

179 CG CAT GGC GAC ATC GCC TGC TAT GAA GCC ATG GTG CTG ATG GCG CAG CCG TTC TCT TAC

P H G D I A C Y E A M V L M A Q P F S Y

238 CGT TAC CCG CTG GTC GAT GGC CAG GGG AAT TGG GGC GCG CCG GAT GAT CCG AAG TCA TT

R Y P L V D G Q G N W G A P D D P K S F

297 C GCG GCG ATG CGT TAT ACC GAA TCC CGC CTG TCC AAA TAC GCC GAG CTG CTG TTA AGC G

A A M R Y T E S R L S K Y A E L L L S

356 AA CTC GGT CAG GGG ACT GCG GAC TGG GTG CCA AAC TTC GAC GGC ACC ATG CAG GAA CC

E L G Q G T A D W V P N F D G T M Q E P

414 G AAA ATG TTA CCG GCG CGT CTG CCG AAC ATC CTG CTG AAC GGC ACC ACC GGT ATT GCG G

K M L P A R L P N I L L N G T T G I A

473 TG GGC ATG GCA ACA GAT ATT CCG CCG CAT AAC CTG CGC GAA GTC GCG AAA GCG GCG A

V G M A T D I P P H N L R E V A K A A

530 TT ACG CTG AT

I T L


resistance determining region (QRDR), without any point mutation in topoisomerase IV

(parC) gene. The translated amino acid sequences of the gene are shown under the


1 ACC GAG CTG TTC CTT GTG GAA GGG GAT TCG GCG GGC GGT TCC GCC AAG CAG GCG CGC GAT

T E L F L V E G D S A G G S A K Q A R D 61 CGC GAA TAT CAG GCG ATC ATG CCG CTC AAA GGT AAG ATC CTT AAC ACC TGG GAG GTC TCT T

R E Y Q A I M P L K G K I L N T W E V S 122 CC GAT GAA GTG CTG GCC TCG CAA GAA GTG CAT GAT ATT TCC GTG GCG ATC GGT ATC GAT CC

S D E V L A S Q E V H D I S V A I G I D P 183 G GAC AGC GAC GAT CTG AGT CAG CTG CGC TAC GGC AAG ATC TGT ATC CTG GCG GAT GCG GA

D S D D L S Q L R Y G K I C I L A D A D 243 C TCC GAT GGT TTG CAT ATC GCT ACT CTG CT

S D G L H I A T L L


resistance determining region (QRDR), without any point mutation in topoisomerase IV

(parE) gene. The translated amino acid sequences of the gene are shown under the


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Fig. 38. SDS-PAGE showing expression of cell envelope proteins of Salmonella isolates.

Lane M: Protein molecular weight marker (PMW-M); Lane 1: S. Newport (SN36); Lane

2: S. Newport (SN71); Lane 3: S. Newport (SN71R); Lanes 4: S. Weltevreden (SW9);

Lane 5: S. Weltevreden (SW30) and Lane 6: S. Weltevreden (SW30R)

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Fig . 39. Epithelial cell invasion of S. Newport after 2 h of infection. Error bars represent

the standard deviation

Fig. 40. Intracellular replication and survival of S. Newport after 16 h of infection

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Fig. 41. Epithelial cell invasion of S. Weltevreden after 2h of infection

Fig. 42. Intracellular replication and survival of S. Weltevreden after 16 h of infection

Results

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4.3.6. Real time PCR and quantification of expression of invasion genes of SPI-1

The relative mRNA expression of the hns and SPI-1 (invA, invH and invF) genes to the

internal control gene, gyrB, was determined to ensure that the measurements were not affected

by analytical procedures, such as RNA extraction efficiency, cDNA synthesis or real time

PCR conditions. The synthesized cDNA by using hexamer primer contained all the 5 genes as

detected by PCR (Fig. 43). 300 nM concentration of each primer was standardized with a

single peak in the melt curve and found to be appropriate for subsequent real time PCR (Figs.

44, 45 and 46). The relative expression levels of all the tested genes are shown in Table 18.

The mRNA expression levels of all the tested genes (hns, invA, invH and invF) were

significantly (P < 0.005) decreased after inducing resistance (SN71R) in S. Newport (by 3.36,

59.72, 10.95 and 176-fold, respectively) compared to the wild type nalidixic acid susceptible

strain (SN71) prior to inducing resistance (Fig. 47). The expression of all the tested genes in

quinolone resistant isolate (SN36) was also markedly lower than those of nalidixic acid

susceptible isolates (by 1.85, 6.57, 2.19 and 17.6 fold, respectively). However, in case of S.

Weltevreden the expression level of hns, invH and invF was significantly (P < 0.005)

increased after inducing resistance (SW30R) (0.84, 0.80 and 0.85-fold, respectively) except

invA, which showed significant reduction compared to the wild nalidixic acid susceptible

strain (SW30) (by 1.41 fold). The expression level hns and invF genes in quinolone resistant

S. Weltevreden (SW9) was higher (by 0.33 and 0.58- fold respectively) and invA and invH

was lower (by 155.4 and 23.9-fold respectively) than those of nalidixic acid-susceptible isolate

(SW30) (Fig. 48).

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Fig. 43. RT-PCR showing expression of three invasion genes (invH, invF and invA) and a

histone-like nucleoid structuring gene (hns). Lane M: 100 bp DNA ladder; Lane 1:

Positive control (genomic DNA); Lane 2 and 4: Negative control (Mock reactions, which

did not contain reverse transcriptase in the RT reaction); Lane 3 and 5: cDNA made

from total RNA isolated from Salmonella. gyrB was a house-keeping gene used as an

internal control to ensure that RNA was present in all samples

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Fig. 44. Melting curve (Tm) analysis of Salmonella hns gene

Fig. 45. Melting curve (Tm) analysis of Salmonella invA gene

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invF invH

gyrB

Fig. 46. Melting curve (Tm) analysis of Salmonella invF, invH and gyrB gene

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Table 18. Relative expression levels of hns, invA, invH and invF genes in quinolone resistant and susceptible isolates

b Ratio of nalidixic acid susceptible to nalidixic acid resistant strains

Salmonella

strains

hns

invA

invH

invF

Relative

expression

Ratiob pvalue Relative

expression


expression


expression

Ratiob pvalue

SN71 1.85±0.185

3.36

<0.005

6.57±0.57

59.72

<0.005

2.19±0.19

10.95

<0.005

17.6±1.26

176

<0.005 SN71R 0.55±0.055 0.11±0.011 0.2±0.02 0.1±0.01

SN36 1±0.01 1±0.01 1±0.01 1±0.01

SW30 0.33±0.0524

0.84

<0.005

155.4±5.5

1.41

<0.005

23.9±2.8

0.80

<0.005

0.58±0.06

0.85

<0.005 SW30R 0.39±0.0413 109.8±3.9 29.6±3.3 0.68±0.04

SW9 1±0.01 1±0.01 1±0.01 1±0.01

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Fig. 47. Relative expression of hns and different invasion genes of SPI-1 of S. Newport.

by qPCR assay in HeLa

Fig. 48. Relative expression of hns and different invasion genes of SPI-1 of S. Wetevreden

by qPCR assay in HeLa

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4.3.7. Cytotoxicity assay

The cytotoxocity detection kit measures lactate dehydrogenase (LDH) release from the

cytosol of damaged HeLa cells into the supernatant (Maldonado et al., 2005; Bhunia et al.,

1994). Lactate dehydrogenase (LDH) is a soluble cytosolic enzyme present in the eukaryotic

cells. Upon cell death, the enzyme LDH releases into culture medium due to damage of

plasma membrane. The increase in the amount of LDH in culture supernatant is proportional

to the number of lysed cells.The cytotoxicity assay was carried out after 4 h of infection of

HeLa cells with Salmonella isolates (ST14028, SN36, SW9, SN71, SW30, SW30R and

SN71R). All the resistant isolates (SN36 and SW9) along with the standard (ST14028) and

one of the sensitive isolate (SW30) showed 100 % cytotoxicity, while SN71 showed 92 %

cytotoxicity and the isolates SW30R and SN71R after inducing resistance showed least

cytotoxicity of 68 % and 65 % respectively (Fig. 49).

Fig. 49. Effect of Cytotoxicity induced by Salmonella serovars on HeLa cells

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4.4. Pulse field gel electrophoresis analysis

Fifty four strains of Salmonella isolated from seafood including S. Typhimurium

ATCC 14028 were used for the PFGE analysis (Table 10). Of the 54 strains, 13 strains of S.

Weltevreden, 9 S. Newport, 11 S. Oslo, 3 S. Typhimurium and one each of S. Aba, S. Virchow

and S. Infantis were found to be typeable on restriction digestion of their chromosomal DNA

with the enzyme XbaI (Figs. 50A and 51A). Similarly, 14 strains of S. Weltevreden, 9 S.

Newport and one S. Infantis was typeable on restriction digestion with the enzyme SpeI (Figs.

50B and 51B). All of S. Bareilly and S. Anatum strains and 2 strains of S. Infantis were

untype-able on restriction digestion with both the enzymes.

The 39 isolates restriction digested using XbaI yielded 20 different profiles, which

were discriminatory (D=0.91) at 60 % similarity value. A dendrogram based on the similarity

value is presented in Figure 52. Resitriction digestion with XbaI yielded 6 to 14 bands with

amplicon size ranging between 48.5 kb to 680 kb. Among the 39 Salmonella isolates 26 could

be clustered into 7 different groups (X1-X7) showing diversity in profiles, while the remaining

13 remained unclustered. The unclustered isolates belonged to the serovar S. Weltevreden

(SW30, SW65 and SW9), S. Newport (SN36, SN37, SN35, SN3 and SN70), S. Virchow

(SV17), S. Typhimurium (ST14028), S. Oslo (SO9), S. Aba (SA74), and S. Infantis (SI73).

Heterogenity was observed for serovars of S. Newport (7 patterns), S. Weltevreden (6

patterns) and S. Typhimurium (2 patterns). Among the 13 isolates of S. Weltevreden 6 isolates

(46.15 %) belonged to the PFGE cluster X1, 2 (15.38 %) each to X2 and X3, while the

remaining three (23.07 %) showed individual distinct patterns. The 9 S. Newport strains were

observed to group into two clusters designated X6 (4 strains) and X7 (4 strains) while the

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remaining 5 remained unclustered at 60 % similarity. Ten among the 11 S. Oslo strains formed

a single group (X4) which was further subclustered in to two, where one subgroup clustered S.

Oslo (2 strains) isolated from oysters and another group form a mixture of S. Oslo isolated

from squids and oysters.. S. Typhimurium grouped in a cluster designated as X5. The single

strain of S. Oslo (SO9) from oyster was clustered with S. Newport (SN3) at 53 % similarity.

The single strains of S. Aba, S. Virchow and S. Infantis was clustered with S. Typhimurium, S.

Weltevreden and S. Newport at a similarity of 55 %, 57 % and 35 % respectively. All the

serovars typed in this study were isolated from a particular seafood source. S. Weltevreden

isolates grouped in cluster X1 were isolated from fish and shrimp; whereas X2 was generated

from serovars isolated from fish. Similarly, the clusters X6 and X7 of S. Newport were

generated from the serovars isolated from clam. The cluster X3, X4 and X5 of S. Weltevreden,

S. Oslo and S. Typhimurium was generated from the serovars isolated from mixed animal

sources of squid/ oyster, oyster/shrimp and fish/clam respectively.

The 24 isolates restriction digested using SpeI yielded 9 different profiles, which were

discriminatory (D=0.90) at 42 % similarity value. A dendrogram based on the similarity value

is presented in Figure 53. Restriction digestion with SpeI yielded 4 to 15 bands with amplicon

sizes ranging from 48.5 kb to 680 kb. Among the 24 Salmonella isolates 22 could be clustered

into 7 different groups (S1-S7) based on their profile diversity, while 2 remained unclustered.

The unclustered isolates belong to the serovar S. Newport (SN3), and S. Infantis (SI73).

Heterogenity was observed within the serovars of S. Weltevreden (4 patterns) and S. Newport

(3 patterns). The 14 isolates of S. Weltevreden were grouped in PFGE clusters, designated S1

(35.71 %), S2 (28.50 %), S6 (21.4 %) and S7 (14.28 %). Eight of the 9 S. Newport strains

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were grouped in clusters designated as S3 (33.33 %), S4 (22.22 %) and S5 (33.33 %) and one

(11.11 %) remained unclustered. The single strain of S. Infantis was clustered with S.

Weltevreden and S. Newport at an average similarity of 30 %. The three clusters (S1, S2 and

S7) of S. Weltevreden was generated from the serovars isolated from mixed seafood sources

except S6, where the cluster was generated from the serovars isolated from single seafood

source such as fish. Similarly, the 3 clusters of S. Newport (S3, S4 and S5) were generated

from the serovars isolated from clam.

Fig. 50. Pulse field gel electrophoresis pattern (PFGE) for S. Weltevreden strains isolated

from seafood. (A) Lane M: Digested chromosomes of Saccharomyces cervisiae; Lane 1-

14: XbaI digested PFGE pattern S. Weltevreden. (B) lane 1-14: SpeI digested PFGE

pattern S. Weltevreden strains

A B

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Fig. 51. Pulse field gel electrophoresis pattern (PFGE) for S. Newport strains isolated

from seafood. (A) Lane M: Digested chromosomes of Saccharomyces cervisiae; Lane 1-

13: XbaI digested PFGE pattern S. Newport. (B) lane 1-13: SpeI digested PFGE pattern

S. Newport strains

A B

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Fig. 52. Dendrogram showing percentage similarity between type-able seafood associated

Salmonella generated from XbaI digested pulse field finger printing. X1-X7 represent the

different PFGE profiles resolved at 60% similarity. Cluster analysis was performed

using the unweighted pair group method with arithmetic mean (UPGMA) method

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Fig. 53. Dendrogram showing percentage similarity between type-able seafood associated

Salmonella generated from SpeI digested PFGE finger printing. S1-S7 represents the

different PFGE profiles resolved at 42 % similarity. Cluster analysis was performed

using the unweighted pair group method with arithmetic mean (UPGMA) method

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