INTRODUCTION
Salmonella has been long recognsized as an important pathogen of
human, animals and poultry. The organism has its global distribution and
usually causes enteric disease in human and animals through ingestion of
contaminated food/feed mostly of origin (Sharma and Singh, 1997). Non-
typhoidal salmonellosis is an important food borne infection with an estimated
incidence of 1.3 billion cases and 3 million deaths all over the world (Thong et
al., 1995).
Enteric Salmonella infection is a global problem both in human beings
and animals. Newborn and young animals commonly suffer from enteric
infection within 15 days of their birth (Kaura et al., 2001). Enteric disorders
that occur at organized animal farms in this country account for 10 to 30% of
annual mortaility and thus are responsible for great economic losses. In human,
diarrhoeal syndrome caused by non-typhoidal salmonella is common in tropical
and developing countries. Available data on food poisoning indicate that
poultry meat is frequently involved in cases of human salmonellosis (Galbraith,
1990).
Although, many of routine epidemiological features viz. host specificity,
sources of infection, the forms of disease manifestation and carrier status of the
organisms have been documented (Wray and Sojka, 1997; Thapliyal, 1979).
Some new epidemiological parameters are being studied in recent years to
elucidate the disease process of salmonellosis. Among these, antimicrogram
1
studies and plasmid profile are of major importance. On study of
epidemiological feature of salmoellosis, plasmids have been reported to play an
important role in virulence and antibiotic resistance of Salmonella (Aarestrup
et al., 1997).
Bacteria are among the chief causal agents of acute diarrhoea and the
majority of the bacterial enteropothogens appear to cause fluid lose by
stimulation enterocytes to actively secrete electrolytes (Rao and Field, 1985).
Elaboration of one or more enterotoxins is recognized as an important
pathological attribute of some non-invasive diarrhoea including bacteria in
elucidating of their enteropoathogenic activity (Rao and Field, 1985; Harne et
al., 1994). Various Salmonella serotypes have been shown to produce
enterotoxins (Baloda et al., 1983; Rahman et al 1991a). The enterotoxic moiety
from salmonella serotypes has been purified and characterized by several
workers (Finkalstein et al., 1987; Rahman et al., 1994). Salmonella
pathogenesis is a complex and multifactorial phenomenon. Salmonella species
interact with ileal mucosa and disrupt normal intestinal functions, which result
in acute inflammatory cell influx, fluid secretion and enteritis (Wood et al.,
1998). Many genes are required for full virulence but only a few of these have
been shown to be necessary for the induction of enteritis (Wallis and Galyov,
2000). Like many other gram-negative pathogenic bacteria, Salmonella
possesses a dedicated protein secretion system denoted as Type III Secretion
System (TTSS), which plays a central role in virulence (Huech, 1998).
Salmonella serovars have been reported to produce several Type III secretions
2
including SIP (Salmonella Invasion Protein) and Sop (Salmonella outer
protein). Of the different Sop proteins, Sop B is associated with enteritis and
diarrhoea. The gene encoding production of Salmonella outer protein (Sop B)
is located on Salmonella specific DNA fragment representing a pathogenicity
island, SPI 5 (Wood et al., 1998) and is found to be widely distributed among
Salmonella (Rahman et al., 2001).
Another important type III secretion is Sop E, which is associated with
invasion by stimulating membrane ruffling (Hard et al., 1998). Unlike Sop B
gene, the Sop E is not widely distributed among Salmonellae and diarrhoea and
as the cases of this kind is frequently observed in livestock and poultry as well
as in human.
Apart from these outer proteins an important pathogenicity is exhibited
by Salmonella species i.e. Salmonella enterotoxin. Although most of the
Salmonella serovars were shown to produce enterotoxins, enterotoxigenicity of
Salmonella has been found difficult to detect by different assays since
Salmonella produce very level of enterotoxin when cultured by conventional
methods (Baloda et al., 1983). Moreover, a major portion of Stn is retained as
cell-bound content, which is not released in the extrcellular milieu (Kaura and
Sharma, 1988). These are some Salmonella strains, which produce enterotoxin
but do not release extracellularly and thus may escape different tests and
detected to be non-enterotoxigenic if only entracellular occurrence of Stn is
tested (Rahman et al., 1991a).
3
Production of the enterotoxin (Stn) has been found to be mediated by the
presence of Stn gene and it has been cloned and sequenced (Chary et al., 1993;
Chopra et al., 1994). More recently, the Polymerase Chain Reaction (PCR) has
been used for the detection of Stn gene (Prager et al., 1995; Rahman, 1999). Its
detection by the PCR in field isolates of various Salmonella can indicate the
protential of Stn production. Use of the standard PCR technique to study the
presence of Stn gene among the field isolates can an important tool in the
molecular characterization of field Salmonella isolates.
The epidemiology and pathogenic process in salmonellosis is dictated
by an array of factors that act in tandem and ultimately manifest into the typical
symptoms of salmonellosis. In recent years, a number of virulence-associated
fimbriae including Salmonella Enteritidis Fimbriae designated as SEF and
Plasmid Encoded fimbriae designated as PEF have been identified and cloned
(Clouthier et al., 1994). However, information on the occurrence of these
fimbriae among the field isolates is still meagre (Rahman et al., 2000a). Both
the above types of fimbriae are genetically encoded and its characterization can
help in understanding the role of these genes in expression of these virulence
factors.
4
AIM AND OBJECTIVES
The aim of the project was undertaken with the following objectives
with given 50 samples:
I. To study the Antimicrogram patterning of the given isolates.
II. To study some of the molecular characteristics of the given
isolates such as
III. Detection of Sop B gene.
IV. Detection of fimbrial genes (Sef and Pef).
V. Detection of salmonella enterotoxin gene (Stn)
VI. Detection of Sop E protein by Dot-ELISA.
VII. Study on plasmid profiles.
5
REVIEW OF LITERATURE
Salmonella
The importance of bacteria of the genus Salmonella as potential
pathogens of human and animals needs no emphasis. Salmonella are widely
distributed in nature. Currently, there are more than 2400 Salmonella serotypes
prevalent in the world. A few of them are highly host specific, while majority
of them are unadapted and can cause infection in a wide variety of animals
species (Gupta and Verma, 1993). They are the chief cause of diarrhoeal
disease all over the world (Gianella, 1980). Newborn and young animals
commonly suffer from enteric infections within 15 days of their birth (Kaura et
al., 2001). Enteric disorders that occur at organized animal farms account for
10 to 30% of annual mortality in our country and are thus responsible for great
economic loss Kaura et al., (2001).
The genus name Salmonella has been adopted in the honor of Salmon,
who isolated the “hog cholera bacillus” considered to be the causal agent of
swine plague (Salmon and smith, 1885) but subsequently the organism was
found to be only a secondary invader and named as Salmonella Choleraesuis.
The typhoid bacillus was first seen by Eberth (1880) in the spleen and
mesenteric gland of patient that died of typhoid and the organism was isolated
by Gaffkey (1884) and Schotmuller (1901) differentiated Salmonella
Paratyphi A and Salmonella Paratyphi B.
6
The genus Salmonella of the Enterobacteriaceae family consists of
Gram-negative bacilli, fermentative, aerobic or facultative anaerobic bacteria
that are generally motile with peritichous flagella except Salmonella pullorum
and Salmonella gallinarum. The organisms of the genus are non-lactose
fermenters, oxidase negative, urease negative acetyl carbinol negative,
potassium cyanide negative but citrate positive (Sleigh and Duguid, 1989).
Nomenclature of Salmonella species
Nomenclature and classification of Salmonella are ever changing. In
recent years, the Kauffman- white Antigenic scheme as proposed by Ewing
(1986) has been modified and updated and this updated version is being
accepted by majority of the laboratories worldwide.The members of of the
genus Salmonella have been grouped in two species, viz. Salmonella bongori
containing 18 serovars and Salmonella enterica consisting of more than 2400
serovars (Popoff and lallinov, 1997). Salmonella enterica has been further
divided into subspecies, viz. Salmonella enterica subsp. arizone (IIIa or 3a),
Salmonella enterica subsp. diarizone (IIIb or 3b), Salmonella Enterica subsp.
houtenae (IV or 4) and Salmonella Enterica subsp. indica (VI or 6). According
to the latest nomenclature, the names of the serovars are not italicized (Popoff
and Lellinov, 1997) and the first letter of the serovar is written in capital hand.
For example, the strain that used to be identified, as Salmonella typhimurium is
now known as Salmonella typhimurium
Antimicrogram patterning
7
Until the fifties, nearly all Salmonella were sensitive to a wide range of
antimicrobial agents. However, due to indiscriminate use of antibiotics
worldwide both in humans and animals as medication or in animals feeds,
plasmid mediated resistance has appeared in them throughout the world. Huey
and Edwards (1958) demonstrated resistance to Tetracyline in Salmonella
strains of Salmonella Typhimurium isolated before 1948, when antibiotics were
not in animal feeds and after 1956 against Tetracyline. It was observed that 5%
of human and 9% of fowl strains exhibited resistance to the antibiotic since the
time Tetracycline was incoprated in the feed. Similarly, Yurack (1964) showed
that 54 of 644 Salmonella cultures exhibited resistance to one or more of
Tetracyline, Chloramlhenicol and Ampicillin. Further, it was found that
Salmonella Typhimurium showed much higher resistance to Chloramphrnicol.
Sojka et al., (1972) made a survey on drug resistance of 2143 Salmonella
strains comprising of 44 serotypes isolated from animals (1329 from cattle, 511
from poultry, 183 from sheep, 74 from pigs and 46 from other species of
animals and miscellaneous sources) in England and Wales. The most common
serotype tested was Salmonella Dublin (925 strains) and Salmonella
Choleraesuis (483 strains). Of these strains, 7.8 % were sensitive to all the
drugs under test. Majority of the strains (91.3%) were resistant to
Sulphadimidine, one strain to Chloramphenicol, 0.2 per cent to Furazolidone,
13.4% to Sulphathiazole, 3.5 percent to Oxytentracycline, 1.2 percent to
Ampicillin, 0.6 % to kanamycin and 0.5 % to polymyxin B. But none of the
strains isolated from poultry was resistant to Streptomycin. In another study,
8
Sojka et al., (1974) isolated Salmonella organisms from animals in England
which showed resistance to Streptomycin (80.9%) Sulphonamide (54.5%),
Ampicillin ((3.7%), Furazolidone (1.7%), Chlortetracycline (1.3%),
Chloramphenicol and Neomycin (0.45 each). However, none of the strains
were resistant to Trimethoprim. Sethi et al., (1976) studied the resistance
pattern of 104 Salmonella strains to Chloramphenicol and reported that strains
isolated from animals and poultry did not develop resistance to
Chloramphenicol in India as compared to some other parts of the world.
In India, Kaliannan and Gupta (1977) studied the drug resistance pattern
of 90 Salmonella strains comprising of 11 serotypes isolated from human and
animals against Chloramphenicol, Tetracycline, Kanamycin, Streptomycin and
Ampicillin and stated that 4 out 10 Chloramphenicol resistant strains were also
resistant to Tetracycline and streptomycin. Mc Garr et al, (1977) studied the
drug sensitivity of 219 Salmonella strains from poultry to 16 antimicrobial
agents. All the strains were sensitive to Ampicillin, Trimethoprim-
Sulphamethoxazole complex, Furazolidone, Cephaloridine, Amoxycillin and
resistant to Tetracycline and Streptomycin. Upadhyay and Misra (1978)
reported that Salmonella Weltevreden isolated from buffalo, goat, pigs and
poultry were sensitive to Ampicillin, Tetracycline, Oxytetracyline,
Streptomycin, Chloramphenicol, Kanamycin and Nitrofurantoin, but were
resistant to Bacitracin. Ishiguro et al., (1979) reported Salmonella strains
resistant to Tetracycline, Streptomycin and sulphadimethoxine from faecal
samples of pigs. Further (Ishiguro et al., 1980) studied that 59% of 84
9
Salmonella isolates were resistant to one or more antibiotics such as
Tetracycline, Streptomycin, Sulphadimethoxine and Cholramphenicol. Anon
(1980) reported resistance of Salmonella to Chloramphenicol, Streptomycin,
Chlortetracyline, Neomycin, Ampicillin and Furazolidone. Gyurov (1980)
demonstrated the drug resistance of 326 Salmonella strains isolated from
poultry. No strain was found to be resistant to Gentamicin, whereas, only one
was resistant to Kanamycin. Of the 326 strains, 1.57% was resisitant to
Carbenicillin, 2.45 percent to Chloramphenicol, 2.76 percent to Streptomycin,
27.6% to Tetracycline and 5.85% to Furazolidone. All were resistant to
Oxacillin and majority to Erythromycin, Penicillin and Ampicillin.
Koshi (1981) reported that strain of Salmonella Typhimurium isolated in
Southern India showed alarming increase in multiple drug-resistances. The
overall drug resistance of Salmonella Typhimurium increased from 59.6 % in
1978 to 90.6 % in 1979 and to 97.1 % in 1980. But the resistance against 5 to 9
drugs (Ampicillin, Chloramphenicol, Gentamicin, Kanamycin, Neomycin,
Streptomycin, Sulfonamide, co-trimoxazole and Tetrcycline) increased
alarmingly from 38.9 percent in 1978 to 83 % in 1979 and 95.5 % in 1980.
Nabburt et al., (1981) reported susceptibility of 333 strains of Salmonella to
eight antimicrobial agents. It was observed that 99.4 % of the strains were
sensitive to Cephalothin, 99 % to Furadione, 98.8 percent to Chloramphenicol,
98.2 % to Ampicillin, 96.7 % to Polymxin, 95.8 % to Kanamycin and 91 % to
Streptomycin. Only 50.4 % of the strains were susceptible to Tetracycline,
whereas 44.1 percent were intermediate sensitive and 5.4 % were resistant to
10
this drug. Gupta et al. (1983) revealed that 44.0 % of 261 Salmonella strains
were resistant to Tetracycline, 9.2 % to Chloramphenicol and 3.8 % to
Ampicillin. In all, 59.7 % and 97.6 % strains were senitive to Chlormphenicol
and Nitrofurantoi, respectively. Hsu et al., (1983) reported that out of 98
isolates of pig including Salmonella Choleraesuis and Salmonella
Typhimurium examined for drug susceptibility, 94 (95.5%) were resistant to
two or more drugs. The percentage of isolates resistant to individual drugs were
as follows: 95.9 percent to Sulphathiazole, 89.8 percent to Tetracycline, 76.5
percent to Streptomycin, 56.1 percent to Kanamycin, 53.1 percent to
Chloramphenicol and 45.9 percent to Ampicillin. However, all the isolates
were sensitive to Colistin, Gentamicin and Nalidixic acid. Blackburn et al.,
(1984) observed multiple resistance in 80 % Salmonella recovered from
animals. The percentage was higher in the isolates from swine. Similarly, Pohl
et al., (1984) found that multiple drug resistance was common in Salmonella
Typhimurium isolates from animals, meat and feeds.
Kim et al., (1984) studied drug sensitivity of Salmonella isolated from
chickens in Korea and reported that 65 isolates were sensitive to Ampicillin,
Colistin, Chloramphenicol, Gentamycin, Kanamycin and Trimethoprim-
Suplhamethoxazole, while 70.8 % were resistant to Streptomycin, 5.39 % to
Sulphonamides, 41.5 % to Tetracycline and 4.6 % to Nitrofurantoin. Siddique
et al., (1985) reported that of 111 strains of salmonella isolated from poultry,
majority showed sensitivity to Ampicillin, Kanamycin. Gentamycin, Neomycin
11
and Streptomycin, while most of them were resistant to Tetracyline, Tylosin,
Trimethoprim, Sulmethoxazole and Furazolidone.
Verma et al., (1993) tested the in-vitro drug sensitivity of 38 Salmonella
Virchow strains identified at the National Salmonella Center. All the strains
were sensitive to Contrimoxazole, 97.37 percent to Chloramphenicol, Nalidixic
acid and Streptomycin. All the strains were resistant to Bacitracin,
Chlortetracycline, Oxytetracycline and Sulphamethoxazole. High levels of
resistance were found to Tetracycline (97.37 %), Penicillin (81.58 %),
Erythromycin (78.94 %), Nitrofurantoin (74.74 %) and high High degree of
resistance was due to extensive use of antibiotics. Borah (1994) tested the in-
vitro drug sensitivity of 19 Salmonella strains and reported that overall
susceptibility of the strains was highest (100 %) to Gentamycin,
Chloramphenicol, Nalidixic acid and Norfloxacin. Less sensitivity was shown
to Doxycycline, Furazolidone, Tetracycline and oxytetracycline, while all the
strains were resistant to Amoxycillin.
Narayanswamy et al. (1996) studied the drug sensitivity of Salmonella
isolated from pigs. Six isolates of Salmonella representing two serotypes, viz.
Salmonella Choleraesuis and Salmonella Enteritidis were tested. All the
isolates were highly sensitive to Chloramphenicol, Gentamycin, Nalidixic acid
Doxycycline; five were sensitive to Sulphadiazine, Nitrofurantoin and three to
Tetracycline, Oxytetracycline and Erythromycin.
12
In Assam, Rahman et al., (1997) studied the drug sensitivity of eight
strains of Salmonella representing two serotypes, viz. Salmonella Gallinarum
(6) and Salmonella Indian (2). All the strains were sensitive to
Chloramphenicol, Gentamycin and Norfloxacin, seven to Nalidixic acid, six
strains each to Trimethoprim, Streptomycin and Kanamycin, and three strains
to Tetracycline.
Plasmid profile
Plasmids are circular DNA molecules, which exist in the bacterial cells
independent to the chromosome. Plasmids have been known to carry genes
which determine a wide range of biological functions and also impart upon
individual organisms certain characteristic features. Due to this, the role of
plasmids in pathogenesis and epidemiology becomes multifactoral. Conferment
of antibiotic resistance is one such feature induced by plasmid genes. Most
antibiotic resistant bacteria isolated from the field are not chromosomal
mutants but carry the resistance genes on plasmids and many of these plasmids
can be transferred, particularly between different genera of enteric bacteria.
Moreover, many of themspecify resistance to more than one drug and in some
cases, to as many as six or seven unrelated drugs (Williams, 1978). Corpet
(1996) has opined that the use of Antimicrobial Growth Promoters (AGPs) in
animals production is fraught with danger as genetic resistance then the
specific AGPs may be carried on the plasmids and may transfer from animals
to man. In addition to its established role in conferment of antibiotic resistance,
plasmids have been known to play an important role in increased virulence of
13
Salmonella.many serotypes including, Samonella Abortusovis, Salmonella
Choleraesuis, Salmonella Dublin, Salmonella Tyhimurium and Salmonella
Enteritidis have been repoted to possess large virulence-associated plasmids
that range in size from 50 kb to 96 kb (Jones et al., 1998, Popoff et al., 1997).
Most field strains of Salmonella are known to carry plasmids differing
in size and number. The use of plasmids profiles has gained increasing
acceptance in recent years as an alternative means for typing Salmonellae.
Plasmids profile however is not always helpful and sometimes the information
derived from the profile is limited. The demonstration that several strains
harbour and indistinguishable plasmid cannot always be taken as evidence of
their relatedness. In an extensive study carried out by Mohan et al., (1995) on
plasmid profile and antimicrogram pattern of Salmonella Typhimurium strains
encountered in different parts of India, they reported that all except two of the
190 strains were plasmid negative. Among the remaining strains the plasmid
profile could be grouped into as many as 52 different profiles. They found the
considerable variation in the plasmid profiles on regional basis with northern,
central and southern region of India showing 24,12 and 16 different plasmid
profiles respectively. They couldn’t establish any comprehensive correlation
between plasmid profiles and also between antibiotic resistant patterns
Salmonella enterotoxin (Stn)
Non-typhoidal salmonellosis, a disease caused by Salmonella other than
Salmonella Typhi, is an important food-borne infection with worldwide
distribution causing gastroenteritis and diarrhoea (Chalker and Blaser, 1988).
14
Until middle of 1960’s endotoxins of Salmonella were incriminated in the
pathogenesis salmonella infections (Kohler and Bohl, 1966). Taylor and
Wilkins (1961) first observed that strains of Salmonella caused fluid
accumulation in the rabbit intestinal loops but they left the Cell-Free Culture
Supernatant (CFCS) unassayed in the test. Gienella et al., (1974) found that
although strains of Salmonella Typhimurium evoked fluid accumulation
observed in the ligated rabbit ileum, their CFCS and its concentrate were
unable to induce fluid secretion. Sakazaki et al., (1974) observed that out of 13
strains, culture filtrates of 11 and only 4 live cultures evoked fluid secretion in
Rabbit Ligated Ileal Loops (RLIL). But reason for the variability remained
unexplained. Koupall and Deibel (1975) demonstrated that oral administration
of enterotoxic factor of Salmonella Enteritidis in infant mice caused fluid loss.
Rahman (1989) tested CFCS, PDP and purified toxin of Salmonella
Typhimurium for the presence of Permeability Factor (PF). Although delayed
PF were present in all the preparations, rapid PF could not be detected in any of
them. Permeability factor along with induration of rabbit skin was observed
with preparations from Salmonella Typhimurium and Salmonella Enteritidis
(Baloda et al., 1983), and Salmonella Weltevreden (Kaura and Sharma, 1983.
Besides indurations, necrosis was also observed in concentrated CFCS of
Salmonella Enteritidis and Salmonella Typhimurium (Baloda et al., 1983) PDP
and purified Stn of Salmonella Typhimurium (Rahman, 1989). The presence of
enterotoxic activity, CHO cell elongation factor, delayed PF in the same peak
(SG-200 FPC) which yielded a sigle protein band in polyacrylamide gel
15
electrophoresis and the observation that these activities were neutralized by the
antiserum to the purified enterotoxin of Salmonella Typhimurium indicated
that the three were due to a single moiety (Rahman et al., 1994).
Detection of Stn gene by PCR
In recent years, nucleic based diagnostic techniques have been very
popular due to their high sensitivity, rapidity and specificity. The same
techniques are now also being introduced for detection of various gene encoded
virulence factor. Production of the Stn has been found to be mediated by the
presence of Stn gene. The chromosomal Stn gene has been cloned and
sequenced (Chary et al., 1993; Chopra et al., 1994). The PCR has been used for
the detection of Stn gene (Prager et al., 1995, Rahman, 1999b). Its detection in
field Salmonella isolates indicates the potential of Stn production. Use of the
standard PCR technique to study on the presence of Stn gene among the field
isolates can be an important tool in the molecular characterization of field
Salmonella isolates. In a study carried out in 46 strains of 2 species of
Salmonella i.e. Salmonella Typhimurium and Salmonella Enterititidis, Rahman
(1999b) observed that Stn gene could be detected with the help of PCR using
known primers amongst all the serovars of Salmonella Typhimurium and
Salmonella Enteritidis whereas the E. coli serovars negative for Stn gene. He
16
also reported that only 34.6% of the Stn positive organisms as detected by PCR
expressed phenotpically in the CHO assay.
Salmonella outer protein (Sop) B and (Sop) E
Salmonella pathogenesis is a complex phenomenon involving several
pathogenic mechanisms. Salmonella sp. interact with illeal mucosa and disrupt
normal intestinal function, which results in an acute inflammatory, cell influx,
secretion and enteritis (Wood et al., 1998). Galyov et al., (1997) reported that
interactions between intestinal epithelial cells and the pathogens play a key in
mediating the inflammatory response. It has been shown that enteropathogenic
Salmonella strains are able to induce intact intestinal epithelia to recruit sub
epithelial neuttrophils ((Mc Cormick et al., 1995a, 1995b). This strains
epithelial signalling to neutrophils requires the adhesion of Salmonella to the
epithelial signalling to neutrophils requires the adhesion of Salmonella to the
epithelial apical membrane and is dependent on protein synthesis in both
bacteria and eukaryotic cell.
Salmonella entry into nonphagocytic epithelial cells requires several
chromosomal genes (inv/spa) clustered in pathogenicity island termed SPI 1
(Salmonella Pathogenicity Island 1). SPI 1 encodes a (TTSS) type III secretion
system, a dedicated protein secretion system of Salmonella, which plays a
central role in the virulence.
17
Salmonella serovars have been reported to produce several type III
secretions including Sip and Sop are found to contribute to virulence by
directing secretion and translocation of several bacterial affector protein into
the cytoplasm of the host cells (Wood et al., 1998).
Sop B (Salmonella outer protein B) a novel enterotoxin of Salmonella
Dublin is a major Sop protein involved in enteropathogenesis and exhibits
extensive sequence similarity to the Ipg D protein of Shogella Flexeri (Galyov
et al.,1997).
Wood et al., (1998) reported characterization of Sop B and presented
evidence that Sop B is translocated into the eulkaryotic cells via, a Sip
dependent mechanism to promote fluid secretion and inflammatory response.
Translocation of intracellular activities of Sop B affects cellular response
leading to influx of neutrophils into the intestinal epithelium and induction of
fluid secretion. Jones et al., (1998) investigated the enteropathogenicity of
another Salmonella Dublin effector protein Sop D. The ability of mutant
Salmonella Dublin strains to induce fluid secretion and influx of
polymorphonuclear leukocytes (PMN) in bovine ligated ileal loops was
investigated. Comparing with the type strain, Sop D mutant Salmonella Dublin
induced less secretion and PMN influx. The Sop B mutant strain also induced
lower secretory and inflammatory responses than the wild type strain. When
both Sop B and Sop D mutations were introduced into Salmonella Dublin, it
resulted in even lower secretory and inflammatory responses than either of the
single mutants. Thus it was suggested that Sop D had a role in the induction of
18
enteritis and that Sop b and Sop D acted in concert to promote fluid secretion
and inflammatory responses in the infected ileum.
Another important type III secretion is Sop E and it is associated with
invasion by stimulating membrane ruffling (Hardt et al., 1998). Unlike Sop B
gene, Sop E is not widely distributed among Salmonellae (Rahman et al.,
2001). The Sop B has been reported in several serovars of Salmonella recently
((Rahman et al., 2001).
Detection of Sop E antigen by serological assay (Dot-ELISA)
The Enzyme Linked Immunosorbent Assay(ELISA) was applied for
assaying cholera toxin for the first time by Holmgren and Svennerholm (1973).
The level of sensitivity of this ELISA technique was calculated to be
approximately 0.09 µg/ml for cholera toxin. Yolken et al., (1977) employed
this technique for detection of E.coli LT enterotoxin. Saxena and Sharma
(1987) also used this test to detect salmonella enterotoxin and they compared it
to that of Rrabbit Ligated Illeal Loop (RLIL) technique.
A different type of ELISA called GM1 ganglioside ELISA was also
widely used for detection of enterotoxin of E.coli (Svennerholm and Holmgren,
1978). The test is based on the fact that ganglioside is a specific receptor of LT
enterotoxin of E.coli. This test is also used for detection of heat stable toxin
(ST) of E. coli and as low as 1 to 2 mg the toxin in crude culture filtrates was
detected (Urban et al., 1990). Dot-ELISA is one of the solid phase
immunoassay developed by Hawkes et al., (1982) to detect either antigen or
19
antibody. Panigrahi et al., (1987) developed an immuno-dot-blot assay for
detection of cholera related enterotoxin of Salmonella Typhimurium where he
used nitrocellulose paper for absorption of the enteroxin instead of polystyrene
plates and they could detect also was 0.02 ng of purified enterotoxin.
Rahman et al., (1991) used ELISA and GM1 ELISA to test for
enterotoxigenicity of Salmonella isolated from food of animals origin. ELISA
could detect as low as 15 ng/100 ml of purified Salmonella enterotoxin and 10
ng/100 ml of cholera toxin when tested with their homogeneous antisera. Gaol
and Liw (19930 identified 329 serotypes of E.coli isolated from diarrhoeic
piglets based on antigenic difference of adhesins by using Dot-ELISA.
Rahman (1999) examined 19 strains of Salmonella enterica belonging to
seven serovars and two strains of Salmonella Bongori for enterotoxin
production by using Chinese Hamster Ovary (CHO) cell assay and Dot-ELISA.
The number of strains, which showed positive reactions in CHO cell assay and
Dot-ELISA, were 13 (64.42 %) and 16 (84.21 %) respectively.
Roychoudhury (1999) studied the production of heat labine enterotoxin
(LT) of E.coli strains using two immunological testes, viz. Biken test and Dot-
ELISA and found that 80.89 % strains were positive for LT in Dot-ELISA,
while 43.82 % were positive in Biken test.
Detection of fimbrial genes (Sef and Pef) by PCR
All members of the family Enterobactericiae express fimbriae, which
allow them to attach to the host surfaces. This attachment is the first step in the
20
colonization of their preferred host niche. Depending upon their
haemagglutinating properties, fimbriae were initially classified into three types:
Type 1 fimbriae were those whose agglutination is prevented by co incubation
with D- Mannose (Old, 1972); Type 2 were the non agglutination types
(Lockmann and Curtiss III, 1992); Type 3 were those which agglutinated with
animal erythrocytes only if they were coated with tannic acid (Duguid et al.,
1996). An additional thin fimbriae with a unique curly morphology in members
of the Enterobacteiaceae has also been described (Arnqvist et al., 1992) and
these show a high degree of sequence conservation. In addition, a fifth
enterobacteial class of fimbriae, termed type 4 fimbriae, has been shown to use
a different export systems for translocation of its fombrial subunits across the
cytoplasmic membrane (Strone and Lory, 1993).
Serovar Enteritidis produces at least five distinct fimbriae, namely
Salmonella Enteritidis Fimbriae (SEF) 14, SEF 17, SEF 21, (all type 1
fimbriae) LPF and PEF (Rajashekara et al., 2000). The information regarding
the role of these fimbriae is still very ambiguous. Of all serovar Enteritidis
fimbriae, SEF 14 has been studied the most. SEF 14 has been known to consist
of 3 major protein subunits sefA (14kd), sefB (28 kd) and sefC (90 kd), which
are arranged sequentially in the fimbrial structure and sefC forms the last and
largest component of fimbriae. sefA,sefB,sefC are encoded by fimbrial operons
sefA, sefB, sefC respectively (Clouthier et al., 1994). Subsequently Clouthier
et al., (1994) also reported one more fimbrial structureb enconded by sefD in
the same 14 fimbrial gene cluster of Salmonella Enteritidis. Amongst the
21
remaining fimbriae, SEF 17 is encoded by agf BAC (Collinnson et al., 1993)
and SEF 21 by fimA (Muller et al., 1991) genes.
Rahman et al., (2000) conducted a PCR amplification study to observe
the occurrence of Sef and Pef genes among different serovars of Salmonella
using specific primers of internal fragments of sefC and pefA fimbrial operons.
They found that strains of Salmonella Enteitidis harboured both sef and pef
genes irrespective of their source. Strains of Salmonella Typhimurium and
Salmonella Gallinarum only per and sef genes respectively. Salmonella
Newport. Salmonella Kentucky, Salmonella Weltevreden and Salmonella
Indiana were found to be negative for both Pef and Sef genes.
22
MATERIALS AND METHODS
BACTERIAL STRAINS
Standard strains
The enterotoxigenic strain of Salmonella Enterica subspecies Enteric
belonging to server Salmonella Typhimurium and Salmonella. Enteritidis were
used for the experimental study. These strains were already serotyped in
National Salmonella and Escherichia Center, Central Research Institude,
Kasauli, Himachal Pradesh (India).
Maintenance of bacterial cultures
All the 50 bacterial cultures were collected from the different sources
such as pig, poultry, cattle, humans and quail by different methods and
maintained through sub-culturing on buffered nutrient agar slants at regular
interval of 3 weeks. The cultures were checked for their purity by streaking on
Brilliant Agar (BGA) and their morphological, cultural and biochemical
characteristics were confirmed (Edwards and Ewing, 1986).
Antimicrogram patterning
In vitro susceptibility of the given Salmonella strains to various
antibiotics was determined by the disc diffusion technique (Kim et al., 1984).
Pure cultures of each isolate was inoculated into 5 ml of nutrient broth and
incubated for 6-8 hrs. Purity of the cultures was checked by microscopic
examination. About 2 ml of each culture was spread uniformly on the surface
nutrient agar (BBL laboratories, USA) plates and kept undisturbed for 5 min.
23
The excess inoculums was sucked out from the plate with the help of sterile
Pasteur pipette. The inoculated plates were allowed to dry in an incubator at 370
C for 5 min. The antibiotic discs were placed gentle on the inoculated agar
surface maintaining adequate distance from each other. The discs were pressed
gentle with the help of a pair of sterilized forceps to ensure complete contact
with the medium. The plates were then incubated at 370C for 18-24 hr. The
zone of inhibition around the discs was measured
PLASMID PROFILE
Extraction of plasmid DNA ( Alkaline Lysis Method)
Extraction of Plasmid DNA was carried out by alkaline lysis method as
per the modified procedure of Kado and Liu (1981) as described by Kinde et
al., (1996). The reagents and buffers required for Plasmid extraction are given
in appendix. A loop full of culture was grown in 3ml of Luria broth (Hi-Media)
and incubated at 370C for 16-18 hr. After overnight incubation, the bacteria
were harvested by centrifugation incubation at 8000rpm for 10 min. The
washed pellet was resuspended in 100μ of (solution I) freshly prepared
lysozyme buffer and incubated at room temperature for 10 min. 200μl of
(solution II) SDS reagent [0.2 N NaOH, 1% Sodium Dodecyl Sulphate (SDS)]
was added to the resuspended bacteria and after gentle shaking, the suspension
was left on ice for 10 min. 100μl of (solution III) (pH 5.6) 3 M potassium
acetate was added to the suspension. The suspension was then vortexed
thoroughly and left on ice for 30 min. The suspension was then centrifuged at
10,000rpm for 10 min at 40. The supernatant was carefully separated and then
24
mixed with equal volume of phenol: chloroform: isoamyl alcohol solution
(25:24:1) and after careful mixing the solution was centrifuged at 10,000rpm
for 10minutes at 40C. The supernatant was removed and left at room
temperature for 5 minutes after thorough mixing. The precipitated DNA was
harvested by centrifugation at 15,000rpm for 10 minutes. The DNA pellet was
then washed with 70% ethanol to remove the isopropanol and air dried for 15
min. The glassy pellet was then resuspended in about 20μ of TE buffer.
Agarose gel electrophoresis of plamid DNA
The plasmid DNA was visualized by carrying out agarose gel
electrophoresis in a horizontal submarine electrophoresis (Pharmacia) as the
procedure described by Sambrock et al., (1989). For electrophoresis, 0.7%
agarose gel in TBE buffer was used. About 7μl of Plasmid DNA mixed with
2μl of gel loading buffer as loaded into the gel with the standard markers.
Electrophoresis was carried out at 80 V for 2 hr minutes till the bromophenol
blue of the gel loading buffer migrated more than fourth and fifth the length of
the gel. At the end of the electrophoresis, the gel was stained with ethidium
bromide (0.5mg/ml) and was visualized in the gel documentation system
(Image Master-VDS, Amersham Pharmacia, Sweden). Molecular weight of the
Plasmid was determined with the help of AAB image software (Amersham
Pharmacia, Sweden).
DETECTION OF SALMONELLA OUTER PROTEIN (SOP) E
Isolation of Sop E secreted by different strains of Salmonella
25
The Sop E was isolated from all the strains of Salmonella as per the
method described by Rahman et al., (2001). Bacteria were grown on Luria
Bertani (LB) agar (Difco, UK) overnight at 370C. One colony from agar plate
was inoculated in 5 ml of Luria Bertani broth (Difco, UK) containing 0.3 M
NaC1 and incubated at 370C for 6 hr (OD,0.7 to 0.8) on a rotary shaker (100
rpm). The culturer was then diluted 4 times in fresh LB broth (final volume 20
ml) in a 100 ml conical flask and incubated at 370C for 18 hr on rotary shaker
(100 rpm). Then the culture was cooled in ice bath for 30 min and centrifuged
at 20,000rpm at 40C for 1 hr. The culture supernatant was collected and filtered
(0.45µm, Sartorius, Goettingen, Germany). The protein present in the
supernatant was precipitated with 10 % (v/v) trichloroaceticacid (Sigma, USA)
for 1 hour and then centrifuged at 20,000rpm at 40C for 1 hr. The sediment was
collected and dissolved in 0.4 ml of NaOH (0.1 M) to which 2.0 ml ice-cold
acetone (- 200C) was added and incubated at - 200C for 20 min. The suspension
was centrifuged at 20,000rpm at 40C for 15 min. The sediments were
redissolved in 20 ml of acetone (- 200C) and incubated and centrifuged as
above. The sediments were dried at room temperature and dissolved in 0.1ml of
PBS and stored at 40C.
DOT-ELISA
The Dot-ELISA for detection of Sop E was carried out as described by
Rahman (1991) with modification. Standard strains of Salmonella Tyhimurium
and Salmonella Enteritidis were used as test isolates a single isolate of E. coli
was used as negative control.
26
Assay Procedure
Two μl of Sop E preparation of 20 randomly selective strains of
Salmonella were dotted on polyvinyldine membrane (PVD) strip (Sigma,
USA), which was prewetted with methanol and dried at 40C for overnight. The
unsaturated sites were blocked by immersing the strips in 3% of bovine serum
albumin, fraction V (Sigma) in PBS for 1 hr at 370C. The strips were washed
three times in wash buffer (0.01 M PBS, pH 7.2 with 0.3% Tween-20 and
0.05% Triton X 100) for 5 minutes. The strips were dipped in the Anti-Sop E
antisera diluted 1: 1000 in wash buffer and incubated at 370C for 1 hr. After
incubation, the strips were washed thrice in wash buffer and incubated with
goat antirabbit IgG horse radish peroxidise ((HRPO) conjugate (Boehringen,
Germany) at a dilution of 1: 1000 for 1 hr at 370C. The strips were then washed
thrice in wash buffer and immersed in freshly prepared substrate solution
(sigma fast-TM DAB/ H2O2, Sigma). Washing the strips in running tap water
stopped the enzymatic reaction and a position reaction was indicated by the
appearance of deep purple dot against a while background within 10-15
seconds.
DETECTION OF VIRULENCE GENES OF SALMONELLA
TYPHIMURIUM AND SALMONELLA ENTERITIDIS BY MULTI PCR
Deterction of Stn gene by PCR
The PCR analysis for the deterction of Stn gene was carried out as per
the method followed by Prager et al., (1995) and Rahman (1999). Bacterial
cells from the overnight cultures were suspended in 350 μl distilled water and
27
boiled at 1000C for 10 min. After boiling, the cell suspension were cooled and
were immediately tasted for Stn gene by PCR analysis. Primers used for PCR
reaction were Stn P1 5-TTG TGT CGC TAT CAC TGG CAA CC-3 (lower
primer) and Stn M13 5- ATT CGT AAC CCG CTC TCG TCC-3 (lower
primer). These primers flank a 617-bp segment in Stn sequence. The PCR
mixture (25 μl) included 12.5μl master mix (QIAGEN) containing 2.5 U Tag
DNA Polymerase, 200μM each of dATP dCTP dTTP dGTP and PCR buffer, 5
μl (lμM) each of upper primer and lower primer and 2.5μl of template DNA
(bacterial cell suspension). PCR incubation was performed in a thermocycler
(Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1 min), primer
annealing (590C for 1 min.) and extension (720C for 1min.) followed by
incubation at 720C for 10 min. A 15 μl aliquot of each primer mixture was
electrophoretically separated on agarose gel (1 % containing 0.5 μl/ml ethidium
bromide, pharmacia) and the PCR produced were visualized in the gel
documentation system (Pharmacia)
Detection of Sef gene by PCR
The PCR analysis for the detection of Sef gene was carried out as per
the methods described by Clouthier et al., (1994) and Rahman et al., (2000).
Bacterial cells from the overnight cultures were suspended in 350μl distilled
water and boiled at 1000C for 10 min. After boiling rhe cell suspension were
cooled on ice and immediately tested for Sef C gene by PCR analysis. Primers
used for the PCR reaction were Sef C 5-GCG AAA ACC AAT GCG ACT
GTA -3 (upper primer) and Sef C 5- CCC ACC AGC AAC ATT A=CAT
28
CCC-3 (lower primer). These primers flank a 1103 bp segment in sef sequence.
The PCR mixture (25 μl) included 12.5 μl master mix (QIAGEN) containing
2.5 U Tag DNA polymerase, 200μM each of dATP, dCTP, dTTP and dGTP
and PCR buffer, 5 μl (1μM) each of upper and lower primers and 2.5μl of
template DNA (bacterial cell suspension). PCR incubation was performed in a
thermocycler (Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1
min.) following by incubation at 720C for 10 min. A 15μl aliquot of each
primer mixture was electrophoretically separated on agarose gel (1 %
containing 0.5μl/ml ethidium bromide, Pharmaca) and the PCR products were
visualized in the gel documentation system (Amersham Pharmacia biotech,
Sweden).
Detection of Pef gene by PCR
The PCR analysis for the detection of Pef gene was carried out as per
the methods described by Baumler and Heffron, (1995) and Rahman et al.,
(2000). Bacterial cells from the overnight cultures were suspended in 350 μl
distilled water and boiled at 1000C for 10 min. After boiling the cell suspension
were cooled on ice and immediately tested for Pef gene by PCR analysis.
Primers used for PCR reactions were Pef A1 5 TGT TTC CGG GCT TGT
GCT -3 Upper primer and Pef A2 5- CAG GCC ATT TGC TGA TTC TTC
C-3 lower primer these primers flank a 700 bp segment in Pef sequence. The
PCR mixture (25μl) included 12.5μl master mix (QIAGEN) containing 2.5 U
Tag DNA polymerase, 200μM each of dATP,dCTP, dTTP and dGTP and PCR
buffer, 5μl (1μM) each of upper and lower primers and 2.5μl of template DNA
29
(bacterial cell suspension). PCR incubation was performed in a thermocycler
(Prkin-Elmer, USA) in 25 cycles of denaturation (940C for 55 sec) followed by
incubation at 720C for 10 min. A 15μl aliquot of each primer mixture was
electrophoretically separated on agarose gel (1%containing 0.5μl/ml ethidium
bromide, pharmacia) and the PCR products were visualized in the gel
documentation system (Amersham Pharmacia biotech, Sweden). Tag DNA
polymerase, 200 μM each of dATP, dCTP, dTTP and dGTP and PCR buffer,
5μl (1μM) each of upper and lower primers and 2.5μl of template DNA
(bacterial cell suspension). PCR incubation was performed in a thermocycler
(Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 55 sec), primer
annealing (550C for 55 sec) and primer extension (720C for 55 sec) followed by
incubation at 720C for 10 min. A 15μl aliquot of each primer mixture was
electrophoretically separated on agarose gel (1% containing 0.5μl/ml rthidium
bromide, Pharmacia) and the PCR products were visualized in the gel
documentation system (Amarsham Pharmacia biotech, Sweden).
Detection of Sop B gene by PCR
The PCR analysis for the detection of Sop B was carried out as per the
method described by Rahman (1999). Bacterial calls from boiled at 1000C for
10 min. After boiling, the cell suspensions were cooled on ice and were
immediately tested for Sop B gene by PCR analysis. Primers used for PCR
30
reaction were Sop P1 5- CAA CCG TTC TGG GTA AAC AAG AC-3 (upper
primer and) and SOP M13 5- AGG ATT GAG CTC CTC TGG CGAT -3
(lower primer). These primers flank a 397-bp segment in Sop sequence. The
PCR mixture (25μl) included 12.5μl master mix (QIAGEN) contatiningb 2.5 U
Tag DNA Polymerase, 200μM each of dATP dCTP dTTP dGTP and PCR
buffer, 5μl (1μM) each of upper primer and lower primer and 2.5μl of template
DNA (bacterial cell suspension). PCR incubation was perfomed in a
thermocycler (Perkin-Elmer, USA) in 25 cycles of denaturation (940C for 1
min.), primer annealing (550C for 1 min) and primer extension (720C 2 min.)
followed by incubation at 720C for 10 min. A 15 μl aliquot of each primer
mixture was electophoretically separated on agarose gel (1 % containing 0.5 μl/
ml ethidium bromuide, Pharmacia) and the PCR products were visualized in
the gel documentation system (Pharmacia).
31
RESULTS
A total number of 50 Salmonella isolates were used for the present
study. They were plated on the BGA medium, which showed pink colour
colonies and were sub cultured in the nutrient agar slants they showed white
translucent colonies.
By Gram staining it was found to be pink colour rods under 100X object
and they were undergone some of the biochemical tests. In which they showed
some of the positive and some of the negative results, according to their
biochemical activity.
A total of 50 Salmonella isolates collect from various sources is given in
Table 1. Among these 50 isolates, 18 are Salmonella Typhimurium and 32 are
Salmonella Enteritidis. All the cultures were tested for their sensitivity to 10
different antibiotics using disc diffusion method and the species-wise results
are present in the Table 2.
The Salmonella strains isolated from animals were found to be resistant
to Ampicilin and were sensitive to Nalidixic acid, Norfloxacin, Streptomycin,
Ciprofloxacin, Chloramphenical, Gentamycin, Cotrimaxazole and tetracycline,
six of which belongs to Salmonella Enteritidis and four to Salomonella
Typhimurium.
Among the antibiotics to which comparatively lower resistance was
observed. The isolates were found to be resistant to Nitrofurantoin (12%)
32
belongs to Salmonella Typhimurium, ampicillin (12%) belongs to Salmonella
Typhimurium and Steptomycin (12%) belongs to Salmonella Typhimurium.
Salmonella strains isolated from community infection showed highest
sensitivity 95% to all antibiotics in which all isolates are Salmonella
Typhimurium. In which, 83% showed sensitive to Nalidixic acid, 100%
Norfloxacin, 100% Streptomycin, 100% Ciproflaxacin, 83% Chloramphenicol,
100% Gentamycin, 100% Contrimaxazole, 100% Tetracycline, 100%
Nitrofurantoin and 83% Ampicillin showed sensitivity. The resistance was
showed least effect (5%) to all isolates. Among 6 isolates one isolate was
found to be resistant to Streptomycin (16.6%). One isolate (16.6%) showed
resistant to Ampicillin and one isolate (16.6%) showed resistant to
Cholramphenicol.
The Salmonella strains isolated from hospital infections showed high
sentitivity (88%) to all antibiotics.In which, 77% of isolates showed sensitivity
to Nalidixic acid, 92% showed to Norfloxacin, 92% showed to Streptomycin,
100% showed to Ciproflaxacin, 92% showed to Chloramphenical, 92% to
Gentamycin, 84% showed to Cotrimaxazole, 84% showed to Tetracycline,
100% showed to Nitrofurantoin and 61% showed to Ampicillin .The
percentage of sensitivity of these antibiotics to both Salmonella Typhimurium
and Salmonella Enteritidis (Table 3).
33
The resistance to these antibiotics is least effect with 12%. Almost all
antibiotics are resistant to some isolates except Ciprofloxacin and
Natrofurantoin.
In overall percentage of sensitive against these antibiotics, it showed
different results. For Nalidixic acid it showed 86% for both Salmonella
Typhimurium and for Co- Ttrymaxazole 91% Tetracycline 91%, Norflaxcin
08%, Ciproflaxacin 100%, Nitrofurantoin 98%, Chloramphenicol 89%,
Ampicillin 52, Stretomycin96% and Gentamycin showed 98% (Table 4).
Plasmid profile study carried out for the randomly selected isolates
revealed a distinctly varied plasmid profile. Plasmid profile analysis of a single
isolate of Salmonella Typhimurium revealed the presence of 4 different
plasmid profiles. And another one only revealed the presence of different
plasmid profiles. A single isolate of Salmonella Typhimurium harboured a 2
different plasmid profiles (Fig. 4).
Dot-ELISA test was carried out with sop E preparation of randomly
selected 19 isolates in which 2 were Salmonella Typhimurium and 17 were
Salmonella Enteritidis as test isolates, the E.coli was used as negative control.
All the 17 test isolates were positive for Sop E, and the Salmonella
Typhimurium showed negative results (Fig. 1).
Among the various known virulence genes of Salmonella Typhimurium
and Salmonella Enteritidis, studies on the detection of enterotoxin gene (Stn),
34
fimbrial gene (Sef and Pef) and Sop B gene were carried out by multiple
polymerase chain reaction (Multi PCR) using their specific primers.
The Stn gene was found to be present in 9 among the 10 isolates (Table
5) in which E. coli 33 was used as negative control. All these 9 positive strains
were found to give 617 bp product in the Stn gene segment. The negative
control of E. coli 33 was found to be negative for Stn gene (Fig. 5).
The Sef gene was found to be present in only 5 isollates (Table 5) in
which one isolate was belongs to Salmonella Typhimurium and four belongs to
Salmonella Erteritidis. All these strains were found to give rise to an 1103 bp
product in the sef gene segment. The Sef gene was found to be absent in the E.
coli 33 strain negative control (Fig. 5).
The Pef gene was found to be present in 4 of the 10 isolates (Table 5).
All of these strains were found to give rise to a 700 bp product in the Pef gene
segment. In 4 Salmonella Typhimurium, 2 showed the presence of this gene
and in 5 Salmonella Enteritidis, 2 showed positive for Pef gene. The control of
the E.coli 33 strain showed negative result (Fig. 5).
The Sop B gene was found to be present in all 9 positive controls (Table
5). of these strains were found to give rise to 317 bp in the Sop gene segment.It
was found to be present in both Salmonella typhimurium and Salmonella
enteritidis isolates and the negative control of E.coli 57 was found to be absent
for Sop B gene (Fig 4).
35
Table 1. Details of samples which were used.
36
Sl. No. Sources of the given samples No. of samples
% of samples
1. Hospital infections 30 60
2. Community infections 10 20
3. Animal infections 10 20
Table 2. Antimicrobial agent their concentration used for antibiogram
studies of the Salmonella isolates.
S.No Antimicrobial Concentration of disc/µg
1. Norfloxacin (Nx) 100
2. Chloramphenicol (C) 80
3. Nalidixic acid (Na) 70
4. Ciprofloxacin (Cf) 60
5. Co-Trimaxazole (Co) 55
6. Tetracycline (Tc) 90
7. Streptomycin (S) 50
8. Nitrofrantoin (NF) 70
9. Ampicillin (Am) 90
10. Gentamycin (Gm) 70
Table 3. Antimicrobial sensitivity pattern of Salmonella Typhimurium
and Salmonella Enteritidis.
S.No Culture Antimicrobial agents (%)
Na Co Tc Nx Cf Nf C Ap S Gm
1. Salmonella 78.5 100 86 100 100 93 78.5 57 93 100
37
Typhimurium
2. Salmonella
Enteritidis
87.5 87.5 94 97 100 100 94 50 97 97
Table 4. Overall percentage of antimicrobial sensitivity pattern of
Salmonella Typhimurium and Salmonella Enteritidis.
38
Table 5. Detection of Virulence genes of Salmonella Typhimurium and
Salmonella Enteritidis.
39
Culture Antimicrobial agents (%)
Na Co Tc Nx Cf Nf C Ap S Gm
Salmonella Typhimurium
and Salmonella Enteritidis
98 91 91 98 10
0
98 89 52 96 98
S.No Virulence
Genes
No.of
Salmonella
Typhimurium/9
isolates
No.of
Salmonella
Enteritidis/9
isolates
Molecular
Weight
1. Stn 4 5 617bp
2. Sef 1 4 1103bp3
3. Pef 2 4 700bp
4. Sop B 4 5 317bp
40
41
42
43
FIG.2 ANTIBIOTIC SENSITIVITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES
44
FIG.3 ANTIBIOTIC SENSITIV ITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES
45
46
47
48
FIG.9 ANTIBIOTIC SENSITIVITIES OF SALMONELLA TYPHIMURIUM & SALMONELLA ENTERITIDIES
DISCUSSION
All the 50 cultures were tested for antimicrobial patterning, with 10
different antimicrobial agents. Gupta et al., (1983) tested 261 Salmonella
isolates with chloramphoenical and they got the result of 91.6% of sensitive but
in the present study showed 97.8% sensitive.
In overall percentage of sensitive to all antimicrobial agents for both
organisms, such as Salmonella Typhimurium and Salmonella Enteritidis, it
showed that 98% sensitive to Nitrofurantoin, Norfloxacin and Streptomycin.
Salmonella Typhimurium showed more sensitive than the Salmonella
Enteritidis. In addition 100% sensitive to Gentamycin and Norfloxacin.
The present study showed very less resistance percentage in both
organisms. Among all the antimicrobial agents Ampicillin showed higher
resistance than the others. Salmonella Enteritidis showed 50% of resistance
against Ampicillin. Especially, poultry isolates showed 92.3% of resistance.
Similar observation done by Blackburn et al.,(1984) reported 80% of resistance
against the culture, isolated from different animals.
Plasmid profile showed different results based on the species and was
found that a single isolate of Salmonella Typhimurium showed high molecular
plasmid. Mohan et al., (1995) reported that except 2 of the 190 strains were
plasmid negative. But the present study showed 100% result for the presence
of plasmid and their different plasmid profiles also i.e. a single isolate of
49
Salmonella Typhimutium showed 4 different plasmid profiles and in
Salmonella Enteritidis also showed 4 different plasmid profiles. It was found
that 3,2 and single plasmid profiles also.
Rahman et al., (1991) examined 29 Salmonella strains for the detection
of enterotoxin and they found 84.21% of positive result by Dot-ELISA. The
present study showed positive result for the presence of Sop E protein in all
the Salmonella Enteritidis isolates and sop E protein is absent in Salmonella
Typhimurium isolates.
Among the various known virulence genes of Salmonella, the detection
of enterotoxin gene (Stn), fimbrial genes (Sef and Pef) and Sop gene were
carried out by multi PCR using their specific primers.
The present study showed that all the isolates of Salmonella Enteritidis
and Salmonella Typhimurium were present in (Salmonella enterootoxin gene)
and found that this gene containing 617 bp by PCR technique. Rahman et al.,
(1991) observed this gene in 58 Salmonella isolates belongs to Salmonella
Typhimurium and Salmonella Enteritidis by the PCR technique. The present
study concludes that the Stn gene contains 617.
Similar type of results were observed by Galyov et al., (1997) observed
that sop B (a 60 Kda protein) encoded sop B genes from a sip B mutant of
Salmonella enterica, and this gene contains 317 bp. The present study is
confirmed that the Sop B gene contain 317 bp with their results.
50
Rahman et al., (2000) conducted a PCR amplification study to observe
the occurrence of Sef and Pef genes among different serovars of Salmonella.
The present study also confirmed that the presence of Sef and Pef genes in
Salmonella Typhimurium and Salmonella Enteritidis and Sef gene is having
1103 bp and Pef gene is having 700 bp.
51
SUMMARY
The present study was aimed to study some of the molecular
characteristics of Salmonella and to evaluate the performance of some of the
available methods for detection of Salmonella enterotoxin (Stn) gene, fimbraial
genes (Sef and Pef), Sop B genes and sop E antigen. Apart from that the
plasmid profile and antibiotic sensitivity were also studied. The evaluation of
the various direct (Dot- ELISA) and indirect methods detection of Salmonella
enterotoxin (Stn) gene by PCR, presence of Stn gene, fimbraial genes (Sef and
Pef and Sop B genes) were carried out in Salmonella Typhimurium and
Salmonella Enteritidis were also studied.
Fifty given isolates which were isolated from various sources, such as
hospital infections, community infections and animals were used for the
present study. These were subjected to in-vitro antibiotic resistance and
plasmid profiling studies.
The antimicrogram revealed varying antimicrobial sensitive patterns.
Most of the antimicrobial agents, which were used, are very effective to all the
isolates especially Norfloxacin, Gentamycin, Nalidixic acid Ciproflaxacin,
Chloramphenicol and Nalidixic acid to be showed a reasonably good efficacy.
On the other hand the highest number of isolates were showed resitance to
Ampicillin and Tetracycline. Salmonella Typhimurium showed higher
sensitivity to most of antibiotics than the Salmonella Enteritidis.
52
Plasmid profile showed a multilateral distribution of plasmid amongst
both serotypes. A total of 2 plasmid pictures were observed. All the isolates
showed different plasmid profiles. Higher plasmid number (three or more) was
observed in both isolates (one one each).
Detection of Salmonella enterotoxin (Stn) was carried out by PCR
(indirect method). Dot-ELISA was found to be the most suitable in terms of its
accuracy, simplicity and rapidity. In the molecular assay, Stn gene was found
to be present in all the 9 isolates that were detected by the presence of a 617 bp
product following PCR with primers. Considering its percent detection level
and this method was found to be most sensitive.
Detection of sop E was carried out by the immunological assay Dot-
ELISA. Twenty isolates were used for this test which 3 isolates belongs to
Salmonella Typhimurium and 17 belongs to Salmonella Enteritidis. Since the
procedure was very easy and it will be more useful for some other
immunological assays.
Detection of fimbrial genes (Sef and Pef) was carried out by PCR using
known primers. Sef gene, detected by the presence of 1103bp product and
found to be present of Salmonella Typhimurium isolate. The Pef gene was
found to be presence in both Salmonella Typhimurium and Salmonella
enteritidis isolates, which was detected by the presence of a 700bp product.
Detection of Sop B was carried out by PCR using known primers. Sop
B, gene detected by the presence of 1348bp product, was found be present in
53
both the isolates of Salmonella Typhimurium and Salmonella Enteritidis.
Considering its percent detection level, this method was found to be most
sensitive. By these detections by PCR amplification technique, it was
concluded that technique is more sensitive since its percent detection level.
54
CONCLUSION
It can thus be concluded that the antimicrogram pattern of the
given 50 salmonella isolates was performed in determine in order to
determine this species type. From the derived antimirogram, it can be
confirmed that the Salmonella Typhimurium isolates are more sensitive
to all the given antimicrobial agents than Salmonella Enteritidis.
Typhoid being the major disease, the antimicrogram helped to
determine the highest sensitive to Gentamycin, Co-Trymaxazole,
Norfloxacin and Ciprofloxacin and it conformed the best applicable
agents for the present day.
This finding was in agreement with the respective plasmid profile
studies; the PCR technique was successfully applied to amplify the
virulence genes (Sef and Pef; Sop B) of both species and its plasmid
profile study.
Identification and determination of various molecular
characteristics of two species enhances the knowledge towards the
vaccine production.
55
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