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Salmonella was first isolated by Salmon (Smith 1894), a pathologist belong to the family Enterobacteriaceae. Salmonella is divided into to species, Salmonella enterica and Salmonella bongori. Salmonella enterica is classified into 6 subspecies, I, II, IIIa, IIIb, IV and VI and Salmonella bongori contains the formerly classified V (Popoff et al. 2004). These are gram negative and non spore forming bacteria. They are facultative anaerobes that produce acid of glucose fermentation and reduce nitrates. Contaminated food and water are the main sources of this bacterium (Szanton 1957; Mead et al. 1999).
Salmonella species often cause specific and non specific characteristic clinical disorders (Saphra and Winter 1957; Rubin and Weinstein 1977) in the patients such as enteric fever, gastroenteritis (Yang et al. 1996), bacteremia, localized and vascular infections (Dhanoa and Fatt 2009). The severity of the infection depends on the Salmonella serotype and various existing conditions of the host, including immunosuppression, vaccinations, previous exposure, age and demographic location.
Acute gastroenteritis with distinct features from that caused by other pathogens results from infection from non- typhoidal Salmonella (Crum 2005) within 6- 48 hours of consumption of Salmonella contaminated food or water. Common symptoms include nausea, vomiting and diarrhoea. In case of most patients with diarrhoea the stools are loose, in moderate volume with no blood (Dhanoa and Fatt 2009). This often lasts for 3- 7 days. Fever 38- 39° C, abdominal cramping, headache and myaglias are other common symptoms in these patients (Hohmann 2001). Patients are relieved of the fever within 48- 72 hours. Hospitalization is often necessary in the affected patients, especially in elderly and in infants due to high risks of dehydration leading to fatality.
Enteric (Typhoid) fever :
Fever and abdominal pain are the typical symptoms of enteric fever of which the causative pathogens are S. typhi and S. paratyphi (Wain et al. 1998; House et al. 2001). The incubation period of these pathogens are 5- 21 days and the diarrhoea lasts for several days. Presence of blood in stools is also a symptom of typhoid fever (Crum 2003). Constipation is another symptom in this case but occurs in a very low population of patients. Chills, diaphoresis, headache of the frontal lobe, weakness, sore throat, muscle pain, cough and dizziness are some of the other non - specific characteristics of this disorder. By the end of first week maculopapular patches appear commonly observed as rose spots on the trunk (Khan et al. 1999). The third and fourth week of this infection may show gastrointestinal bleeding and intestinal perforation in certain cases due to ulceration and necrosis at the primary site of infection (Bhutta 2006).
Bacteremia and vascular infection:
Non typhoidal gastroenteritis is usually followed by bacteremia and some of these cases end up in localised infections. These are common in cases of S.choleraesius and S.dublin. Endovascular infections are uncommon complications of salmonellosis and are not detected till later stages of the infections.There is a high risk of endovascular infections being complicated by bacteremia (Hsu et al. 2003).
Other localised infections:
Extraintestinal localised infections in the bone, central nervous system, muscle and soft tissues, spleen, urinary and genital tract have been associated with salmonellosis. Localised infections in the femour, tibia, humerus and lumbar vertebrae that can be diagnosed with the help of a bone radiograph are rare and non fatal. Osteomyeletis and joint destruction of the hip, knee and shoulder is another low fatality salmonellosis infection. Meningitis, ventricultis and encephalopathy leading to seizures, mental retardation and brain infarction are infections localised to the central nervous system. Cerebrospinal fluid culture and MRI scan helps diagnose this condition. Septic thrombophlebitis and endophthalmitis of the soft tissues can be diagnosed using drainage cultures. Abscesses of the genito- urinary tract are common in elderly female patients and pregnant women (Sulaiman and Sarwari 2007). The incidence of this infection is rare and has low mortality rates. This is often diagnosed using ultrasonography, urine cultures if infection is in the urinary tract and aspiration. Splenomegaly occurrences have also been rare but are yet another manifestation of salmonellosis (Khan et al. 1999).
Laboratory diagnosis of salmonellosis is done on the basis of microscopic examination of stools collected from the patient. In case of gastroenteritis, microscopic examination of stools reveals presence of neutrophils and at times even red blood cells (Crum 2003). A confirmation on the diagnosis can be made after the isolation of Salmonella species from the blood, rose spots of the trunk, bone marrow, stools, and intestinal fluids from the infected patient. In case of enteric fever routine examination of the bone marrow is recommended and is more favourable than a blood culture examination. Duodenal secretions are examined using the duodenal string test. This test has a high sensitivity and is non- invasive. Stool cultures give positive results in the third week of infection if they have been given no therapy thus far. The bacteria is isolated using selective media , Hektoen Agar and Salmonella- Shigella agar (Crum 2003). Hence the diagnosis of enteric fever in patients can be confirmed by examination of the blood, bone marrow and intestinal secretions, including duodenal fluids and stools. Serological tests to detect antigen or antibody to confirm presence of S.typhi are also followed but the sensitivity of these tests has often been questioned. Widal test is an example (Khan et al. 1998). Rapid analysis kits those are commercially available for serological testing is replacing these tests in most places due to its greater sensitivity and better performance. Various other PCR assays and DNA probe assays have been developed for the detection of S.typhi but they are not usually employed in clinical environments. Bacteraemia is diagnosed by culturing blood from patients onto sheep blood agar and gram staining single colonies (Graham et al.). Antibiotic susceptibility tests are done using disc diffusion technique to study the resistance of the strains (Graham et al. ; Wain et al. 1998).
Shigellosis or bacillary dysentery is a result of infection of Shigella organisms, non motile and non encapsulated gram negative bacilli of the family Enterobacteriaceae (Watson et al. 2006 ). It was only at end of 19th century was a line drawn between bacillary dysentery and amoebiasis, the two forms of dysentery. The 47 serotypes of Shigella are categorized into 4 serologically similar groups, group A (S.dysenteriae), group B( S.flexneri), group C ( S.boydii) and group D (S. sonnei). The most common pathogen and highest number of cases of dysentery are reported to be caused by S.sonnei. This bacillus is shown to produce toxins which play an important role in its pathogenicity. Toxin sequences have been identified in 80% of the strains of the Shigella species. ShET1 and ShET2 are two of the enterotoxins carried by this species. Shigellosis is a person to person transmitted disease and occurs more in crowded environments which has low standards of hygiene with food and water being the most common vectors for this bacteria (Collier et al. 1998a; Mandell 2009).
The characteristic symptoms of shigellosis or bacillary dysentery are blood in the stools, mucoid stools or watery diarrhoea along with abdominal pain (Bern et al. 1992). These symptoms are although very inconsistent in an infected population (Mata et al. 1970). Specificity of the combination of these symptoms varies. Fever is another symptom commonly observed in these patients which can go as high as 105 °F. The initial symptoms of infection include fever and abdominal cramps along with watery loose stool (Taylor et al. 1988; Kosek et al. 2003). This is followed by a drop in fever and cramping and increased passage of stools of small volume (Ferreccio et al. 1991). The site of infection is the colon. Blood or mucus in the stools is observed at this stage of infection (Speelman et al. 1987). Abdominal tenderness over the lower abdominal region and bowel sounds are also observed on physical examination. Rectal mucosa ulcerations are common and are observed after a few days of infection. Reactive arthritis and haemolytic - uraemia syndrome are abnormalities that occur from infections with S.dysenteriae and S.flexneri. studies conducted have shown that the Shigella bacillus starts multiplying within 12 hours of ingestion by the host bowel to high concentrations of 108 viable cells/mL at which point the abdominal cramps and fever begin in the patient. Other symptoms that are observed in these patients include urgency and tenesmus which occur during latter stages of infection (Collier et al. 1998a). The symptoms appear on the basis of the colonic localization of the bacteria in the host. Abdominal pain gets confined to the lower abdominal region along with tenderness of the abdomen (Echeverria et al. 1991).
Shigellosis is suspected in cases of patients having diarrhoea with bloody or mucoid stools and fever and further laboaratory diagnosis is done to confirm the presence of the pathogens (Speelman et al. 1987). This is essential because dysentery is a symptom in case of infection with Entamoeba histolytica, Salmonella spp, Campylobacter spp and Yersinia enterocolitica. Fecal blood in case of Shigella is found to be bright red unlike the other pathogens. Also on microscopic examination of stool samples reveals sheets of polymorphonuclear leukocytes, PMNs. More than 50 PMNs per high power microscopic field is usually observed and just 10 leukocytes are enough to confirm presence of Shigella (Perdomo et al. 1994; Mandic-Mulec et al. 1997). During the latter stages of infection the PMNs degenerate and hence this diagnostic test would lose its importance. Identification of Shigella from the stool cultures after isolation would be a definite clinical diagnostic test (Speelman et al. 1987). Rectal swabs are also employed for isolation of Shigellae. These swabs are advantageous over stool cultures as stool cultures have to be processed immediately whereas swabs can be stored overnight at 4°C. For storage purposes the swabs are suspended in phosphate buffered glycerol saline holding solution. Differential media and selective media, MacConkey agar, hektoen enteric agar and Salmonella- Shigella agar are used to isolate the bacteria. These media allow the growth of Shigella as they contain bile salts to prevent growth of gram positive species and lactose fermentors from the non- fermentors would be differentiated with the help of pH indicators. Secondary isolation media, such as Kligler's iron agar (KIA) and Triple sugar iron (TSI) are employed after overnight incubation at 37 °C of the primary media to stab individual non lactose fermentor colonies. Acid butt and alkaline slant are indicative of presence of Shigella species. Further serological tests are done to identify the serotype such as the agglutination tests. PCR is employed for detection of Shigella species from stool cultures (Taylor et al. 1988; Dutta et al. 2001)and has been found to be a sensitive and rapid test to confirm the presence of Shigella virulence plasmids using primers specific to the plasmids (Pal et al. 1997; Mandell 2009).
Campylobacteriosis refers to infections caused from the bacteria Campylobacter sp. Campylobacter is a motile gram negative comma shaped bacilli. Although this bacteria is known to cause diarrheal and systemic illness not all of the Campylobacter sp produce illness (Collier et al. 1998b). Campylobacter sp are microaerophilic and 5- 10% oxygen is best suited for their growth and the best suited temperature is 42°C. The C.jejuni is has an enormous serotypic diversity with more than 90 different serotypes. This bacterium does not survive freezing or drying temperatures, standard chlorine levels in water and pasteurization. The large reservoir of Campylobacter sp in animals is usually responsible for infections in humans through contaminated meat or other animal products or even direct contact (Coker et al. 2002). Person to person transfer of infection is also common in this case of bacteria. Human bile helps in the multiplication and colonization of Campylobacter, upper regions of the small intestine thereby becoming the primary site of infection. The flagellae of the bacteria is known to have virulence properties as it promotes motility which assists in colonization of C.jejuni. The bacteria are also known to produce proteins and a cytolethal distending toxin but their roles have not yet been defined although they have been found to cause cell death. Lipopolysaccharides with endotoxis activity have been found in the outer membrane of Campylobacter. C.jejuni and C.fetus are two most common infection causing strain of Campylobacter (Blaser et al. 1980; Mandell et al. 2009).
Almost all species of Campylobacter spp that cause enteric illness cause identical clinical manifestation and hence C.jejuni can be used as a prototype for he same. The most common presentation would acute enteritis with symtoms lasting from a day to a week. The other associated symptoms include headache, malaise, myaglia lasting 12- 24 hours (Collier et al. 1998b). This is followed by intestinal illness. Diarrhoea and abdominal pain are the most common and severe symptoms (Allos 2001). Diarrhoea in this case could be anything from loose stools to watery stools to bloody stools (Bokkenheuser et al. 1979). Abdominal pain or rather abdominal cramping is relieved by defectaion and most patients have upto 10 bowel movements on the worst day of the infection. Some patients also experience delirium and rigors, febrile seizures or convulsions are common in children (Coker et al. 2002). Abdominal pain is very severe in case of Campylobacter infections compared to other enteropathogens. The other manifestation of C.jejuni would be acute colitis. Symptoms include low grade fever, diarrhoea and abdominal cramps. Diarrhoea lasts upto a week and worsens with passage of blood in the stools as the infection progesses (Eberhart-Phillips et al. 1997). Another common symptom of this manifestation is tenesmus. In some cases of infection, abdominal pain in the right lower quadrant of the abdomen would be the only presenting symptom in the patient. septic abortion is at time acaused due to an existing (Coker et al. 2002)infection in the pregnant patient. Extraintestinal manifestations of the infection include skin lesions, osteomyelitis, reactive arthritis and prolonged rheumatic symptoms after several weeks on the infection.
C.fetus infections differ from C.jejuni infections significantly. C.fetus produces systemic symptoms usually and is an un common diarrheal illness pathogen. Intermittent diarrhoea or abdominal pain is similar to C.jejuni infection although they are uncommon (Coker et al. 2002). A prolonged relapse is a characteristic of C.fetus with fever, chills and myaglia. Other extraintestinal sites of infection include vascular sites causing necrosis in endocarditis and pericarditis patients. Bacteremia is common and develops as thrombophlebitis (Guerrant et al. 1979). Also in pregnant women, infections develop with symptoms of bacteremia and fever. Infections from C.fetus Subsp fetus are fatal in immunocompromised patients.
Campylobacter infection is established using laboratory to confirm its presence either by direst examination of faeces or by isolating the bacteria (Allos 2001). Serological tests are also done for research purposes. The diarrheal faecal samples are collected from the patient and are examined using microscopic techniques, namely darkfield or phase contrast (Bokkenheuser et al. 1979). The examination is done within two hours of passage of stools and the diagnosis is established on the basis of the motility. They are found to show rapid jerking motion in wet preparations and they have spiral morphology (Sacks et al. 1986). Gram staining is also done to establish the presence of the bacteria. In stained smears they are identified because of its spiral morphology (Sacks et al. 1986). In case of Campylobacter enteritis infection the faeces are also examined for the presence of red blood cells and neutrophils using direct microscopy. PCR and FISH techniques have also been employed to detect Campylobacter in the isolates. FISH technique employs fluorescent labelled probes to specific target regions on the ribosomal RNA which allows rapid detection of Campylobacter sp. Selective media is used for the isolation of Campylobacter such as Skirrow's (Moore and Madden 1998), Butzler's, Campy-BAP (Bolton et al. 1983). These three are the most commonly used media for this purpose. Filtration techniques are also used for the isolation of Campylobacter using filters with pores of diameter 0.45 µm to 0.65 µm (Engberg et al. 2000). These bacteria are very small (0.3 µm to 0.6 µm in diameter) will pass through these filters. Colonies appear on plating on selective media with 1 to 2 days. They are identifies on the basis of their staining properties. Young cultures show typical vibriod colonies which which on a 48 hr incubation period appear coccoid. PCR based techniques using specific primers have been developed for confirmation of strains (Winters and Slavik 1995; Ng et al. 1997) and serotyping (Black et al. 1988). ELISA and immunoblotting are also used to identify the antigen present in the strain (Mitchell et al. 1988; Mandell et al. 2009).