V. DICUSSION

Page 144

Foodborne infection due to Salmonella species are a cause of concern worldwide.

Besides vibrios, sporadic incidences of Salmonella related gastroenteritis have also been

reported due to the consumption of sewage contaminated shellfish (Allen, 1899; Greenwood et

al., 1998; Mead et al., 1999). The emergence of antibiotic resistant isolates of Salmonella is a

global concern and has been attributed to the indiscriminate use of antibiotics in humans and

animals. There are a number of reports on serovars such as Salmonella Typhimurium and

Salmonella Enteritidis that are commonly associated with foodborne infections. The

prevalence of ACSSuT phenotype (resistance to ampicillin, chloramphenicol, streptomycin,

sulfamethoxazole and tetracycline) of S. Typhimurium is reported to be on the rise (DeToro et

al., 2011). Several studies have been conducted on antibiotic resistance of Salmonella from

poultry and meat; however, studies on Salmonella from seafood are limited. Many studies

have also identified and characterized class 1 integron of Salmonella from various sources

(Kim et al., 2007; Murphy et al., 2007; Rayamajhi et al., 2008; Zhang et al., 2009; Melendez

et al., 2010; Wannaprasat et al., 2011), but the reports on class 1 integrons of Salmonella

isolates from seafood are very limited.

In South east Asia, S. Weltevreden has been isolated from seafood (Reilly and Twiddy

1992; Shabarinath et al., 2007; Kumar et al., 2009) and is recognized as an important

nontyphoidal serovar causing human infections (WHO, 2005). The limited reports available

on its antimicrobial resistance indicate that it is sensitive to antibiotics (Aarestrup et al., 2003).

Most studies on antibiotic resistance genes are carried out on strains showing phenotypic

resistance to a particular antibiotic. However, during the last decade, there have been reports

of unexpressed antibiotic resistance genes in some bacteria, for example, mecA gene in

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Staphylococcus sciuri (Wu et al., 2001) and cat9b gene in superintegron present in Vibrio

cholerae (Rowe-Magnus et al., 2002). Aquatic systems represent an extraordinary settlement

for the mixing and spread of bacteria with resistance genes and consequently for the horizontal

transference of genetic elements conferring resistance. The association of antimicrobial

resistance determinants with transferable elements may promote the rapid dissemination of

antibiotic resistance among Salmonella serovars. Class 1 integrons containing variety of

resistance gene cassette could play an important role in the dissemination and maintenance of

antibiotic resistance in Salmonella isolates in both the presence and absence of selective

pressure. Thus, analysis of the presence of antibiotic resistance genes and phenotypic

expression of resistance in the seafood associated serotypes of Salmonella are necessary.

Apart from the study of antibiotic resistance pattern it is also important to understand the

variation in the expression of virulence genes of SPIs, growth, infectivity and cytotoxicity of

antibiotic resistant as well as sensitive strains. Further, because of the emergence Salmonella

infection from different food sources it is also important to elucidate the clonal relationship of

different Salmonella serovars isolated from seafood using pulse field gel electrophoresis

(PFGE).

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

The majority of the previous studies on the antibiotic resistance of Salmonella were

conducted on isolates from various foods, but there are very few reports from seafood. It was

observed that the frequency of multiple antibiotic resistance among the seafood isolates of

Salmonella is moderately high (25 %) compared with what has been reported from other

foods. An earlier work (White et al., 2001) reported that 84 % and 53 % of the Salmonella

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isolated from retail meats were resistant to at least one and three antimicrobials, respectively.

A Spanish study (Cruchaga et al., 2001) found that the resistance pattern was similar for both

strains isolated from humans and food, pointing the fact that food is one of the important

means of spreading multidrug resistant Salmonella. Hence, our result highlighting moderately

high antimicrobial resistance in seafood associated Salmonella assumes special significance.

Limited earlier reports on the antibiotic resistance of S. Weltevreden show that the frequency

in this serotype was low. One report hypothesizes that this serotype may not easily acquire

resistance or that their natural reservoirs are not exposed to large amount of antibiotics and

this may be the reason for the low frequency of resistance in the case of S. Weltevreden

(Aarestrup et al., 2003). Our results on the antibiogram of S. Weltevreden show that the

frequency of resistance is not very low. Four of 17 isolates (23.5 %) of S. Weltevreden tested

showed multidrug resistance as against 9.5 % reported earlier (Aarestrup et al., 2003). This

result assumes significance especially when this particular serotype has been recognized as

important cause of nontyphoidal salmonellosis in the Southeast Asian region (World Health

Organisation (WHO, 2005). Salmonella Weltevreden was also reported as the most important

seafood associated serotype in India (Shabarinath et al., 2007). The presence of five

tetracycline resistant genes and one chloramphenicol resistant gene in all the isolates were

tested by PCR. 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 determinant. The gene tetA encodes for membrane associated efflux protein

containing 12 transmembrane segments. The tetracycline resistant isolates contained the tetA

gene, and the gene was located on the plasmid which is in agreement with the earlier report

(Michael et al., 2006a). The gene tetA is identical to the recent reports on S. Typhimurium

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(AB576781), S. Enteritidis (FN995455) and S. Schwarzengrund (CP001125) and other

Salmonella serovars (EU219534, AB366441, AM746674, CP000604 and AY463797),

Escherichia coli (GU371928, GQ214053 and GQ149344), Edwardsiella tarda (CP001136)

and V. cholerae (AB114188). Earlier reports suggest that most frequent types of genes coding

for tetracycline resistance (tet) found in Salmonella belonged to the classes of A, B, C, D and

G. Of this, tetG is present in the Salmonella Genomic island I. The Salmonella strains in the

present study were therefore screened for the presence of other tetracycline resistant genes

such as tetB, tetC, tetD and tetG. Two of our isolates SW9 and SN36 contained tetB and tetG

tetracycline resistant genes, respectively. It is interesting to note that none of the tetracycline

resistant isolates contained more than one tet genes except SW9. Therefore, it is possible that

presence of single tetracycline resistant gene is enough to cause phenotypic resistance

characteristics in Salmonella isolates (Jun et al., 2010). Chloramphenicol resistance can due to

degradation of the antibiotic by either chloramphenicol acetyl transferase or chloramphenicol

efflux mechanism. Tn-9-borne catA1 has been earlier reported in various serotypes of

Salmonella (Kobayashi et al., 2007). The catA gene has been reported to be located on

plasmid pHCMI (Michael et al., 2006a). Likewise, our isolates also contained catA1 in the

plasmid. The gene catA1 reported in this study is identical to that reported from E. coli

(FN554766) and other Salmonella serovars. In the case of S. Weltevreden, one study had

reported that 12.5 % of the tested Salmonella isolates contained catA1 gene (Cruchaga et al.,

2001), whereas in the present study, more than half of the isolates (57.5%) contained catA1

gene. Interestingly, in this study, it was found that 16 chloramphenicol sensitive isolates

possessed catA1 genes. It is possible that the gene is not expressed in these strains. The catA1

gene of sensitive strain (SW30) was 99 % similar with that of resistant strain (SW9). The

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reason for the unexpressive characteristic of the catA1 gene of SW30 in this report was found

to be the deletion of the promoter region which has been confirmed by PCR using cat F2 ⁄ R2

primers. A 528 bp PCR product was obtained in SW9, and no amplification was obtained in

SW30. This study demonstrates for the first time that environmental nontyphoidal Salmonella

strains may be carrying silent antibiotic resistant genes. Silencing of antibiotic resistant genes

in environments, where the gene product does not confer any selective advantage, may be a

phenomenon that has not received much attention. As in this report, the deletion of promoter

region may be one of the reason for silencing a gene, but Enne et al., (2006) reported silencing

of several antibiotic resistance genes such as blaOXA-2, aadA1, sul1 and tetA carried on plasmid

pVE46 in a porcine isolate of E. coli following oral inoculation of organic piglets. The genes

and their promoters were intact, and the silencing of the plasmid borne resistance gene was

because of a chromosomal effect, because the transfer of plasmid to another host led to

expression of resistance in the new host. Even in the original bacterial host, the silencing was

reversed at a low frequency of 10-6

-10-10

. It has been hypothesized that there may be a

reservoir of unexpressed resistance genes in environment bacteria with various environmental

and genetic factors are among the reasons for their unexpressive characteristics. Further, our

isolates also contained integrons that play very important role in acquiring antibiotic resistance

genes. Chen et al., (2004) identified six integron replicons in multidrug resistant Salmonella

from meat. The replicons were sized as 0.75, 1.0, 1.20, 1.50, 2.0 and 2.70 kb long. The most

important antibiotic resistant genes carried by these integrons were aadA1 and aadA2

conferring resistance to streptomycin and dhfrXII conferring resistance to trimethoprim. The

present study demonstrated that 1597 bp sized integron of S. Weltevreden (SW9) contained a

dihydrofolate reductase gene (dhfrA7) and a dihydropteroate synthetase gene along with the

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usual quaternary ammonium compound resistance gene similar to the reports of Tamang et al.,

(2007). The gene cassette showed 100 % identity with dihydrofolate reductase (DHFR) and 99

% identity with dihydropteroate synthetase gene encoding resistance to co-trimoxazole

(trimethoprim + sulfamethoxazole) and sulphonamide, respectively. The cassette array is

identical to that reported from S. Paratyphi A (AM412236), S. Enteritidis (FN995455), E. coli

(X58425, EU935739, EU687490, EU598449, AM886293 and GQ402463). Similarly, our

report also demonstrated that 2055 bp sequenced region of integron of S. Newport SN36

contained dihydrofolate reductase type 1 (dhfrA1), OrfC, quaternary ammonium compound

resistance and dihydropteroate synthetase gene. The genes in the cassette showed 100 %

identity with dihydrofolate reductase type 1 (dhfrA1), OrfC, Orf3 ⁄ QacEdelta1 fusion protein

and with sulphonamide resistant protein. The cassette array is identical to that reported from

Salmonella enterica serovar Emek (AY963803), Acinetobacter baumannii (CU459141 and

CT025832) and S. enterica serovar Albany (AY146989). Thus, the resistance to co-

trimoxazole (trimethoprim + sulfamethoxazole) and sulphonamide was found to be integron

mediated in seafood isolate of S. Weltevreden and S. Newport. The variation in sizes of the

integron between SW9 and SN36 and SN33 (1597 bp for SW9 and 2055 bp for SN36 and

SN33, as shown in Fig. 16A) indicates the presence of more than one gene in case of S.

Newport (SN36 and SN33) when compared with the S. Weltevreden, as shown in the integron

map (Figs. 19A and B). This shows that there is a good chance of more drug resistance genes

or genes of unknown function getting integrated into S. Weltevreden as a result of frequent

dissemination of antibiotic resistance among the serovars. The multidrug resistance

mechanism of Salmonella is highly complicated, and mainly involves plasmids, transposons,

as well as integrons and gene cassette mediated resistance. These integrons are found to be

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located on plasmid which is consider to be a main mechanism for the rapid spread of

multidrug resistance among Gram negative bacteria (Leverstein-Van et al., 2002; Rowe-

Magnus et al., 2002). Earlier reports confirm the presence of class 1 integrons in Salmonella

(Orman et al., 2002; Ploy et al., 2003). However, Aarestrup et al., (2003) could not find

integrons in the environmental isolates of S. Weltevreden. Recently, the presence of class 1

integron carrying dhfrXII and aadA2 genes in Salmonella Lansing and dhfrA1 and orfC in S.

Newport strains from seafood has been well described by Khan et al., (2009), which are

similar to our finding in the case of S. Newport, but this is the first report of the presence of

integron in S. Weltevreden. The presence of integrons in S. enterica serovars is of great

significance, because the strains might easily become resistant to broad spectrum of antibiotics

which remains the basis of therapy against antibiotic resistant and multidrug resistant strains.

In conclusion, this study demonstrates that integrons have been playing an important role in

the development of multidrug resistance in most frequent serovars like S. Newport and S.

Weltevreden in recent times. Analysis of the class 1 integrons revealed the presence of new

variants of resistance genes so far not detected in S. Weltevreden from seafood. Asymptomatic

carriers like seafood may further lead to the dissemination of Salmonella strains not only to

other animals, but also to humans when they enter the food chain. However, the increase in the

number of multidrug resistant Salmonella strains from seafood appears to be an emerging

problem. A better understanding of the molecular mechanisms by which antimicrobial

resistance emerges and spreads should enable us to design intervention strategies to reduce its

progression.

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

genes of SPI- 2

5.2.1. Multiplex PCR (mPCR) assay

Multiplex PCR is a commonly used method for the detection of foodborne pathogen

and is being increasingly applied in diagnostics (Lei et al., 2008; Oh et al., 2009). In this

study, a specific mPCR to detect multidrug resistant Salmonella from seafood in a single step

has been developed and evaluated. All Salmonella strains listed in Table 2 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 florenfenicol. One isolate of S. Newport which was resistant to

sulphonamide was also resistant to tetracycline and florenfenicol thus conferring the isolate as

multidrug resistant (Table 3). Remaining isolates though negative for antibiotic resistance

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

gene operon, invA is essential for the virulence seen in Salmonella and is thought to trigger the

internalization required for invasion into deeper tissues (Galan et al., 1989). All isolates were

positive for the invA gene by PCR confirms them as Salmonella and is in agreement to the

report of Swamy et al., (1996). Resistance to tetracycline and chloramphenicol as noted by the

presence of tetG and florR gene is similar to the results of Cabrera et al., (2006), where most

of the S. Typhimurium strains contained florR and tetG genes. Resistance to sulphonamide

was common in poultry and pork samples with sul gene being generally associated with

integrons (Antunes et al., 2006). Results of our previous study on Salmonella showed sul1

genes to be integron associated (Deekshit et al., 2012). The genes sseF and ssaT coding for the

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effector and apparatus proteins of SPI-2, are important for the intracellular survival of the

organism (Yoon et al., 2009). Several studies have looked upon the development of mPCR for

the detection of different serovars of Salmonella or different genes from different sources

(Alvarez et al., 2004;Teh et al., 2008; Lee et al., 2009; de Freitas et al., 2010; Ngan et al.,

2010; Salem et al., 2010; Akiba et al., 2011; Park et al., 2011; Pui et al., 2011; Wang and Suo,

2011; Jeyasekaran et al., 2012; Liu et al., 2012; Yang et al., 2012). Few authors have

developed mPCR for the detection of multidrug resistant Salmonella (Khan et al., 2000; Ng et

al., 2001; Chiu et al., 2006). However, this study targets the simultaneous detection of genes

of SPI-2 and antibiotic resistance genes from seafood.

5.2.2. Sensitivity of mPCR

The efficacy of mPCR in the identification of pathogens from clinical, environmental

and food samples has been well documented (Mason et al., 2001; Kong et al., 2002; Wang et

al., 2002). 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 4). 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 only after 8 h incubation

with 10-1

(9.83×107CFU/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

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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

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

detection efficiency of multidrug resistant Salmonella. The method developed proved to be

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. The reason for difference in the detection level of Salmonella spp. in

different samples need further study. Thus, the present study could detect Salmonella at lower

inoculum level when compared to the reports of Fach et al., (1999), where a PCR based

method could detect Salmonella only after 18 h enrichment period with 106 CFU per 25 g of

food samples. Several studies have been carried out to detect Salmonella by uniplex and/or

mPCR at different time intervals (Myint et al., 2006; Moganedi et al., 2007 Kumar et al.,

2008b). However, this appears to be the first report on the development of mPCR for

simultaneous detection of SPI-2 of Salmonella and its antibiotic resistance genes from

seafood.

When tested on tenfold serially diluted bacterial genomic DNA, the limit of detection

of the mPCR assay for S. enterica serovars Newport was 1 ng/µL of original dilution (Fig.

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21), which was comparatively more sensitive than the reports of Lim et al., (2003), where 500

ng/tube was the detectable limit for S. Typhimurium.

5.2.3. 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 1). 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.

5.2.4. Detection efficiency of the mPCR

In terms of detection speed of multiple genes, the entire process of the mPCR assay

from sample enrichment to data analysis can be completed in 24 h. Considering together with

the simultaneous identification of antibiotic resistance genes, SPI-2 genes and invasion gene,

the effectiveness of the mPCR was significantly improved from conventional detection and

immunological methods which require 5-7 days for identification (Jasson et al., 2010). Hence,

this mPCR can therefore be used as a rapid and novel method for the simultaneous detection

of multidrug resistant Salmonella serovars along with SPI-2 genes.

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5.3. 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

5.3.1. Role of gyrA mutations in quinolone resistance genotype of Salmonella

Quinolone resistance has increased drastically over the last few years among

Salmonella isolates (Liu et al., 2005). The most important mechanisms producing nalidixic

acid resistance are point mutations in the gyrA gene and overexpression of the efflux pumps.

Reports suggest that single gyrA mutation was sufficient to cause high level of quinolone

resistance in Salmonella (Giraud et al., 2006). However, overexpression of the efflux pumps

were also found to be the widespread mechanism and generally represent the first step in the

acquisition of fluoroquinolne resistance (Giraud et al., 2000). DNA gyrase is a bacterial type

II topoisomerase and introduces negative supercoils into DNA influencing the transcription

process and topoisomerase IV responsible for unlinking the structure formed between two

newly replicated DNA strands during replication process. The mutations in the QRDR regions

prevent antimicrobial agents from binding to their topoisomerase targets (gyrA, gyrB, parC

and parE) and carrying out their antimicrobial activity (Heisig, 1993). In this study the isolates

resistant to nalidixic acid (SN36, SW9 and SN71R) had a single mutation at different positions

in the gyrA region except one of the experimentally selected S. Weltevreden (SW30R), which

did not harbor any mutation in the gyrA, gyrB, parC and parE region even after inducing

resistance, instead the resistance here was thought to be primarily due to other possible means

such as decreased permeability or increased expression of the efflux pumps (Piddock et al.,

1995; Martinez et al., 1998) which is confirmed by the efflux pump inhibition test. In the

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Page 156

presence of efflux pump inhibitor PAβN, the isolates which were completely resistant to

nalidixic acid after inducing resistance showed MIC of 48 µg /ml (SW30R) and 24 µg /ml

(SN71R). According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the

breakpoint of nalidixic acid MIC is established as 16 μg /ml, below this value is included in

susceptible group. However, in our study efflux pump inhibition test using PAβN could not

reduce MICs of nalidixic acid below the CLSI breakpoint. This is in agreement with the study

of Saenz et al., (2004), where the presence of PAβN could not change the nalidixic acid

resistance phenotype in E. coli isolates with mutations in gyrA and parC. This suggests that

QRDR mutation has an important influence on nalidixic acid resistance though efflux pump

was inhibited. However, Saenz et al., (2004) described that gyrA with single amino acid

change required an additional resistance mechanism, such as efflux pump to show a resistance

phenotype against nalidixic acid in E. coli, whereas in our study all of the tested nalidixic acid

resistant Salmonella isolates with a single mutation in gyrA gene showed resistance

characteristics without efflux pump overexpression. To date six major groups of active drug

efflux pump transporters have been identified in prokaryotes: ATP-binding cassette (ABC),

major facilitator superfamily (MFS), small multidrug resistance (SMR), multi-antimicrobial

resistance (MAR), resistance nodulation division (RND), and multidrug and toxic compound

extrusion (MATE) (Paulsen et al., 2003; Van Bambeke et al., 2000). The role of

overexpression of the AcrAB-TolC efflux pump has been well studied in quinolone resistant

Salmonella (Chu et al., 2005; Chen et al., 2007; Fabrega et al., 2009). But in the present study

no resistance was observed due to overexpression of efflux pumps. This has been further

proved by cell envelope protein gel electropheris of the Salmonella isolates where all the

isolates showed similar expression of cell envelope protiens. The mutations found (Ser83 to

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Page 157

Tyr, Asp87 to Asn and Asp87 to Gly) in the QRDR region were always single and had already

been described by other authors (Brown et al., 1996; Griggs et al., 1996; Giraud et al., 1999;

Molbak et al., 1999; Walker et al., 2001). In the present study mutations in Gly81 (Giraud et

al., 1999), Ala119 (Griggs et al., 1996), Ser83 and Asp87-Tyr (Hamidiana et al., 2011) were

not observed. The double mutation in gyrA (both 83Ser and 87Asp) was found to be associated

with resistance to ciprofloxacin at breakpoint concentration of the CLSI (MIC of 32µg/ml)

(Hirose et al., 2003; Capoor et al., 2009), but double mutation was not found in the present

study. Resistance to quinolones can also be mediated by chromosomal mutations in the

subunit B of DNA gyrase and topoisomerase IV, in their QRDRs. However, none of the

identified mutations (Tyr42Cys and Arg437Leu in gyrB; Glu453Gly, Ser458Pro, His461Tyr,

Ala498Thr, and Val512Gly in parE) (Ling et al., 2003; Eaves et al., 2004) were observed in

the target genes of any of the isolates tested in the present study.

Plasmid mediated resistance genes of qnr (qnrA, qnrB, qnrS and qnrD) and aac(6ꞌ)-Ib-

cr has also been described in quinolone resistant nontyphoidal Salmonella (Gay et al., 2006;

Chen et al., 2007; Xia et al., 2009). However in the present study none of the isolates

possessed plasmid mediated quinolone resistant genes such as qnrA, qnrB, qnrS and aac(6′)-

Ib-cr although the plasmid mediated quinolone efflux pump (qepA), was present in the tested

isolates. However, there have been several recent reports of foodborne Salmonella enterica

harboring quinolone resistance genes, qnrS and qnrB2 from chicken meat, carcasses, and

minced meat (Kehrenberg et al., 2006; Avsaroglu et al., 2007; Sjolund-Karlsson et al., 2010;

Fortini et al., 2011; Lee et al., 2012). These reports show the future risk associated with the

world wide emergence of plasmid borne resistant of foodborne pathogens. The earlier findings

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Page 158

confirmed that PMQR genes were located in conjugative plasmids and they may play a role in

the spread of fluoroquinolone resistance through the food chain. Therefore, despite the fact

that no PMQR was observed in this study, frequent monitoring is essential for all resistance

determinants in relation to the severity of the risk in foods. Prevalence of qepA was found to

be apparently low in the previous studies (Poirel et al., 2008) whereas in this study all the

nalidixic acid sensitive strains also contained qepA gene but these plasmid encoded genes did

not show any significant change in their MICs when compared with the isolates susceptible to

nalidixic acid and negative for the presence of qepA. However, aac(6′)-Ib-cr was not detected

in any of the tested isolates. Since the strain SW30R did not contain any mutation in the

QRDR region (gyA, gyrB, parC and parE), harbor PMQR genes and no overexpression of the

efflux pumps, the resistance mechanism in that particular (SW30R) strain is still unclear.The

possibility of having other means of resistance mechanisms apart from those which are

discussed above cannot be ignored. Unknown resistance mechanisms may also play different

roles in the process of acquisition of quinolone resistance. However, mutation in QRDR

region in SN71R is the main reason for nalidixic acid resistance.

Several studies have suggested that, in Salmonella spp., the first and essential step

towards quinolone resistance phenotype is the acquisition of mutations that gives rise to an

increased efflux, mainly due to AcrAB overexpression, whereas mutations in the QRDRs

represent the second step as well as other mutations enhancing the efflux activity (Giraud et

al., 1999; Baucheron et al., 2004; Chen et al., 2007). Whilst in the present study mutation in

the QRDRs represents the essential step in the acquisition of quinolone resistnace rather than

overexpression of efflux pumps. However, according to Fabrega et al. ( 2009) the first step

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Page 159

would be attributed to the implication of an efflux pump followed by target gene mutations as

well as enhanced efflux activity.

This study also showed decreased growth, invasion and intracellular replication of

nalidixic acid resistant isolates, compared to the susceptible isolates. The mechanisms of

altered growth rate are unclear, but the decreased growth may be the result of multiple gene

rearrangements and/or mutations of topoisomerase genes and 16S rDNA in the quinolone

resistant Salmonella strains. However, growth rate is also depends on the rate of transcription

regulated in accordance with physiological demands (Bremer and Dennis, 1996). In

cytotoxicity assay only the isolates after inducing resistance showed least cytotoxicity.

5.3.2. mRNA expression of SPI-1 genes

In this study, it was found that the mRNA expression of 3 SPI-1 genes, (invA, invH and

invF) and a histone like nucleoid structuring gene (hns) was decreased in nalidixic acid

resistant and experimentally selected nalidixic acid resistant S. Newport (SN71R), compared

to quinolone susceptible strains (SN71). invF is a regulatory gene and required for efficient

invasion of cultured epithelial cells (Kaniga et al., 1994), suggesting that it is important for the

expression of other genes required for invasion. invH is a structural gene involved in the

ability to attach to and invade cultured epithelial cells (Altmeyer et al., 1993). invA, a

structural gene, form a channel in the inner membrane of the cell wall through which the

exported polypeptides are translocated which allows the internalization of S. Typhimurium in

cultured epithelial cells (Galan et al., 1989; Swamy et al., 1996). According to Soto et al.

(2006) the frequent use of antibiotics like quinolones can lead to the reduced bacterial

virulence as well as partial or total loss of pathogenicity islands (PAIs) in uropathogenic

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Escherichia coli (UPEC) strains. However, in the present study no loss of virulence genes was

discovered in the quinolone resistant (SN36 and SW9) or susceptible strains (SN71 and

SW30). Instead, quinolone resistant strains (SN36, SW9 and SN71R), but not quinolone

susceptible strains, had a single mutation in the gyrase gene (gyrA). DNA gyrase is a bacterial

type II topoisomerase and introduces negative supercoils into DNA, influencing the

transcription process and, therefore, virulence gene expression (Galan and Curtiss, 1990;

Heddle et al., 2001; Rhen and Dorman, 2005). The decreased mRNA expression of hns, invA,

invH and invF in quinolone resistant strains may have resulted from DNA gyrase gene

mutation, although mutations of other genes of the bacterial genome may have also

contributed to decreased expression of the virulence genes. However, S. Weltevreden, which

had no mutation in the gyrA region even after inducing resistance (SW30R) did not show any

significant decrease in the expression levels of invasion genes (except for the gene invA) when

compared to the sensitive one (SW30). But the mRNA expression levels of hns and invasion

genes of wild resistant isolate of S. Weltevreden (SW9) was reduced significantly compared to

the sensitive strain (SW30). This suggests that though the isolate is resistant to nalidixic acid,

mutations in the QRDR region play a vital role in the expression of their virulence gene.

SPI-1 is associated with invasion of host cell and induction of macrophage apoptosis

(Amavisit et al., 2003) and decreased expression of invasion genes in Salmonella is associated

with reduced invasion in to host cells (Lee and Falkow, 1990; Rhen and Dorman, 2005).

Similarly, in this study the decreased epithelial invasion combined with intra epithelial cell

replication of quinolone resistant Salmonella strains might be associated with decreased

expression of SPI-1 genes. The decreased virulence gene expression, invasiveness and growth

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rates suggest that pathogenicity of quinolone resistant Salmonella might be attenuated.

However, whether quinolone resistant Salmonella is less pathogenic than quinolone

susceptible strains, the exact molecular mechanisms need further study. In conclusion,

quinolone resistance of Salmonella in this study is associated with decreased expression of hns

and SPI-1 virulence genes (invA, invH and invF), slow growth, cytotoxicity and reduced

invasiveness into mammalian cells. The altered expression was not due to mutation of the

virulence genes, but may be related to mutations of other genes, such as the gyrase gyrA gene.

In wild quinolone resistant isolates (SN36 and SW9) the decreased growth rate, invasion and

survival inside the HeLa cell may also be due to other factors such as presence of class 1

integron or resistant to other antibiotics as observed in our previous study (Deekshit et al.,

2012). Molecular mechanism of how particular mutations in the QRDR region (gyrA, gyrB,

parC and parE) improve or decrease the growth rate or expression of particular virulence

associated genes of SPIs are the studies involving future scope. However, many authors have

suggested that specific mutation in the topoisomerase II (gyrA and gyrB) and topoisomerase

IV (parC and parE), possibly by acting in combination to influence the level of superhelicity

in DNA, restore appropriate levels of gene expression at some loci where the loss of MarR

regulation has a negative impact on growth rate (Nollmann et al., 2007; Deibler et al., 2001).

Similarly, Marcusson et al., (2009) identified acquisition of a fourth resistance mutation in the

QRDR significantly increased fitness in vitro and in vivo in low fitness triple mutants while at

the same time dramatically decreasing drug susceptibility. Many studies have shown the

mechanism of quinolone resistance in Salmonella (Ouabdesselam et al., 1995; Eaves et al.,

2004; Solnik-Isaac et al., 2007; Lunn et al., 2010). However, very few studies have

emphasized on the growth, infectivity, cytotoxicity and difference in the expression of

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virulence genes of SPI-1 of quinolone (Nalidixic acid) resistant and susceptible Salmonella

strains (Wang et al., 2009). In general this is the first report on nalidixic acid resistance S.

Weltevreden and S. Newport carrying mutation in QRDR region isolated from seafood.

5.4. Pulse field gel electrophoresis

The incidences and severity of diseases caused by Salmonella have increased in recent

years and several studies have reported an alarming increase in the isolation of resistant

Salmonella from human and non human sources (Brisabois et al., 1997; Kalender et al., 2009;

Oloya et al., 2009). Many DNA based genotyping techniques have been utilized in looking at

the epidemiological relationships between various strains of Salmonella isolated from

different sources (Kostman et al., 1992; Millemann et al., 1996; Tsen et al., 2000), of which

PFGE is currently considered to be the “gold standard” (Soto et al., 2001; Swaminathan et al.,

2001; Cerro et al., 2003; Gatto et al., 2006; Oliveira et al., 2009). Till date several

epidemiological studies have been carried out for different serovars isolated from different

sources using PFGE (Laconcha et al., 2000; Martinez-Urtaza et al., 2005; Seo et al., 2006;

Aktas et al., 2007; Bolton et al., 2007; Willford et al., 2007; Xia et al., 2009; Benacer et al.,

2010; Oliveira et al., 2010; Chen et al., 2011; Zheng et al., 2011). A few studies have also

used different enzymes for evaluating the epidemiological relationships using PFGE (Pang et

al., 2007; Rivoal et al., 2009; Trujillo et al., 2011; Zheng et al., 2011). Since, PFGE studies on

Salmonella serovars isolated from seafood is limited, in this study the genetic diversity that

exists among seafood isolates of Salmonella serovars by PFGE was attempted. Two restriction

enzymes XbaI and SpeI were used for the efficient differentiation of 54 Salmonella strains

from seafood by PFGE. The ability of two enzymes was successfully employed to

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discriminate between 39 (XbaI) and 24 (SpeI) of 54 strains analyzed. The DI value for PFGE

obtained by using XbaI and SpeI restriction enzymes were 0.91 and 0.90, respectively (> 0.90)

which is the acceptable confidence value for understanding the degree of discrimination

(Khoodoo et al., 2002; Kumar et al., 2008b). Both XbaI and SpeI PFGE generated 7 different

patterns which enabled the seafood isolates to be grouped and indicated the diversity of

Salmonella serotypes in this region. Though some of the serovars were untype-able by using

SpeI enzyme, 7 different restriction patterns were observed in S. Weltevreden and S. Newport.

High genetic diversity and limited genetic similarity were observed for XbaI digested S.

Newport serovars whereas, high genetic similarity was found for SpeI digested S. Newport

serovars. The PFGE method was found to be highly discriminatory in this study for subtyping

S. Weltevreden and S. Newport which is in agreement with the earlier studies, wherein a high

discrimination was observed with subtyping of S. Typhi and S. Typhimurium (Thong et al.,

1996; Tsen et al., 2001; Gorman and Adley, 2004). This is evident from the six DNA banding

patterns generated after restriction digestion of 13 strains of S. Weltevreden by XbaI, four

banding patterns by SpeI of S. Weltevreden and 7 and 3 banding patterns upon restriction

digestion by XbaI and SpeI respectively of 9 S. Newport strains. The SpeI digested S. Newport

was linked to a particular seafood type, the clams and the clusters were clearly separated from

the serovar (SN3) isolated from oyster. These results are in agreement with the report of

Bhowmick et al., (2012) where RAPD (Random amplification of polymorphic DNA) was

used as a main fingerprinting technique for the differentiation of Salmonella from seafood.

The cluster X3 and X4 of XbaI PFGE pattern (Fig. 52) contains strains which appear

genetically near similar to one another. Thus, even though these isolates are from different

seafood sources (shrimp/oyster for X3 cluster and squid/oyster for X4 cluster); they could be

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clonally related. Similarly, the serovar S. Oslo originating from squid and oyster of cluster X4

are genetically similar, though they were isolated from different sources, suggesting that there

is movement within the seafood population or dissemination owing to a vector or vectors. The

XbaI PFGE analysis used in this study could also be able to distinguish multidrug resistant

strains (SN36 and SW9) from the sensitive strains (SN71 and SW30) as they shared different

pulsotypes. Genetic diversity between the isolates from different seafood sources would be

common, but the genetic diversity among the similar seafood sources suggests the presence of

different clones of Salmonella which further, increases the risk of seafood being a potential

source of highly pathogenic bacteria like Salmonella. Regardless, these data confirm the

observation that multiple clones of S. Weltevreden, S. Newport and S. Oslo are present in the

environment along south west coast of India. However, horizontal gene transfer is now widely

accepted as an important factor in driving genome composition among enteric bacteria (Brown

et al., 2003), and both diversity and similarity in the genome have been noted as outcomes of

the lateral transfer of DNA among closely related strains (Brown et al., 2001; Dykhuizen et

al., 1991).

In conclusion, PFGE is the widely used molecular tool to determine the genomic

diversity/similarity among Salmonella isolates. It provides a powerful tool for the

epidemiological typing/fingerprinting of different Salmonella serovars isolated from seafood.

Subtyping of Salmonella by PFGE can also be extremely useful when assessing contamination

and dissemination problems in different environments apart from epidemiological

investigations. Molecular fingerprinting evidence by PFGE carried out in this study showed

high diversity among the Salmonella serovars isolated from seafood. Ingestion of

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contaminated seafood could potentially contribute to foodborne salmonellosis in humans,

which calls for hygienic handling by personnel and hygienic standards of the waters from

where seafood is harvested and post harvest handling practices to be taken care of.