Salmonellosis is a group of clinical patterns of infection caused by an approximately 2000 serotypes of Salmonellae tribe. This includes typhoid fever caused by Salmonella typhi, gastroenteritis caused by S. enteritidis and typhimurium, and paratyphoid fever caused by S. paratyphi A and S.schottmuelleri. Salmonellae tribe includes the most important genus Salmonella.The most important pathogen in this genus is S. typhi. The other members are divided into two groups: S. choleraesuis, a zoonosis of swine, and S. enteritiditis, a large super species that is comprising of approximately 1700 serotypes.Each serotype has a species designation that reflects its source, such as S. typhimurium (isolated from rats and mice) and S. durban (the city of isolation).
CELLULAR MORPHOLOGY
The members of Salmonellae are Gram-negative short bacilli that occur singly. They are motile with the help of peritrichous flagella. They are noncapsulated and nonsporing.
CULTURAL CHARACTERISTICS
PHYSICAL REQUIREMENTS
The salmonellae are aerobic or facultative anaerobes. They grow best at 10 to 42°C, optimum being 37°C. They prefer to grow at pH 7 or slightly alkaline pH.
NUTRITIONAL REQUIREMENTS
The salmonellae can grow on simple laboratory media. Various differential and selective media are now available for the growth and isolation of these bacteria.
COLONIAL MORPHOLOGY
The colonies produced by the Salmonella on nutrient agar or other laboratory media are round, 2-3 mm in diameter, convex, smooth, with entire margin, and translucent. They produce diffuse turbidity with powdery deposits in broths.
BIOCHEMlCAL CHARACTERISTICS
Biochemical properties are very important in identification of the members of Salmonella. One of the important properties is their inability to ferment lactose, sucrose, and salicin.
ANTIGENIC STRUCTURE
The salmonellae contain three major antigens: H, or flagellar Antigen; 0, or somatic antigen; and Vi antigen (possessed by only a few serotypes).
H ANTIGEN
H (from German hauch, meaning spreading) antigen is found in the flagella. It is protein in nature. A species or type produces flagella with different chemical organization at different time. This change is called phase variation. H antigen may occur in either or both of two forms called phase 1 and phase 2. The antigen in phase 1 types is specific and found only in few types and reacts with specific antisera. Contrary to this, antigen in phase 2 types is nonspecific and is distributed in a wide range of the salmonellae. It can cross-react with nonspecific antisera. Analysis of H antigen provides a useful epidemiologicai tool to investigate outbreaks of salmonellosis and determine the infection’s source and mode of spread.
O ANTIGEN
0 (from ohne hauch, meaning nonspreading) antigen occurs on the surface of the outer membrane of the bacterial cell. It is also called somatic antigen. Chemically it is the O-specific polysaccharide of the lipopolysaccharide (LPS) component of the cell wall that is liberated on lysis of the cell and, to some extent, during culture. The ilpopolysaccharide moiety may function as an endotoxin and may be important in determining the virulence of the salmonellae.
Lipopolysaccharide complex consists of three components:
* 0 polysaccharide is an outermost coat. The sequence of the sugars monomers in this structure is responsible for 0 antigen specificity. It may also be at determining factor of the organism’s virulence. The salmonellae lacking the complete sequence of O-sugar repeat monomers are called rough because they produce rough colonies. Rough types are usually avirulent or less virulent than smooth types that possess a compete sequence of O-sugars in their LPS. They are called smooth because they produce smooth colonies.
* R core is a middle portion of LPS. Antibodies produced against the R core may protect against infection with a wide variety of Gram-negative enteric bacteria sharing a common core structure.
* Lipid A is the inner coat of the molecule. It is the toxic component of the LPS complex. It is similar in all salmonellae as well as in other members of Enterobacteriaceae. It plays an important role in the pathogenesis of many clinical manifestations, evokes fever, activates the serum complement,
kinin, and clotting systems, depresses myocardial function, and alter lymphocyte function.
Vi ANTIGEN The Vi antigen, also referred to K antigen, is a superficial antigen overlying the 0 antigen. It is polysaccharide in nature and constitutes a component of the capsule (K, from the Danish Kapsel, capsule). It should be mentioned here that the capsule of the salmonellae, as well as of other enteric bacteria, is not very prominent and
well defined. It provides resistance to phagocytic ingestion because of its ability to resist activation of the alternate complement pathway. The Vi antigen is present in a few serotypes of the satmonellae, the most important being S.typhi.
ANTIGENIC CLASSIFICATION
Kauffmann and White have classified the salmonellae on the basis of their antigenic structure. According to their scheme the genus Salmonella is classified into more than 2000 “species” as each antigenic type that was identified was designated a species name, such as S. typhimurium and S. schottmuelleri. Ewing and his associates opposed this classification and presented a different classification containing only three species: S. typhi, S. enteritidis, and S. choleraesuis. All other species or serotypes were defined as serotypes of S. enteritidis. Ewing classification is quite complicated and this is why it is not used in most part of
the world.
RESISTANCE TO PHYSICAL AND CHEMICAL AGENTS
The salmonellae are less resistant to various physical and chemical agents. They are highly susceptible to moist and dry heat. They show high degree of tolerance to relatively larger concentrations of bile. This property has been employed in designing selective media for the isolation of salmonellae by the addition of bile salts. Desiccation is also harmful for them. The salmonellae are susceptible to disinfectants, such as phenol, chlorine, and formaldehyde. Acids and bases can kill these bacteria.
PATHOGENESIS
Salmonellosis is manifested in several syndromes (gastroenteritis, enteric fever, septicemia, focal infection, and carrier state). Particular serotypes show a strong propensity to produce one or the other of these syndromes. S. typhi, S. paratyphi A, and S. schottmuelleri are responsible for enteric fever, S. choleraesuis causes local infections, and S. enteritidis gastroenteritis.
The organisms enter the human body when contaminated food and water are ingested. The patients and carriers discharge these pathogens in their fecal material. Fecal contaminated food and water are the best source of infection. Fingers and flies also play important role in the dissemination of the pathogens. They colonize the ileum and colon, invade the intestinal epithelium, and then grow and increase their population within the epithelium and lymphoid follicles.
The severity of the infection is dependent on certain factors. These include size of the inoculum, the serotype, and the state of health of the host. It has been stated that more serious disease occurs in individuals with debilitating illness. The size of the inoculum depends on the type of serotype and host factors. It has been estimated that as few as 17 or as high as 1 x 109 living cells are required to cause the disease
CLINICAL MANIFESTATIONS GASTEROENTERITIS
The salmonellae invade the intestinal epithelium and cause an inflammatory response in the lamina propria of the intestinal villi. The host’s defense in the area may prevent less virulent serotypes from finding their way into the blood circulation. This keeps the pathogens localized in the intestinal tract. The incubation period of the illness is 6 to 72 hours, usually 12 to 36 hours. The small and large intestines secrete fluid and electrolytes, resulting diarrhea. The mechanism of diarrhea is not very clear but it is believed that it is somehow similar to that caused by V. cholerae. Invasion of the intestinal mucosa is followed by activation of mucosal adenylate cyclase. This resu|ts in the formation of adenosine 3’5'-cyclic phosphate (CAMP). It induces hypersecretion of fluid.
Diarrhea lasts for 2 to 3 days and is usually accompanied by malaise, nausea, and headache. Many infected persons remain asymptomatic.
TYPHOID FEVER
The only etiological agent of typhoid fever is S. typhi. The incubation period is 10 to 14 days, but varying from 5 to 35 days. The bacilli survive and multiply within macrophages in intestinal lymph nodes. These macrophages transport the bacilli to the mesenteric lymph nodes from where they travel to thoracic duct. Finally the bacilli are discharged into blood. This results in massive dissemination of bacilli to various organs including spleen, liver, Peyer’s patches and kidneys. The typhoid bacilli find their way into gall bladder through circulation or biliary tract. Consequently, they enter the intestine for the second time in overwhelming numbers. The result of this second invasion is very serious, leading to a very acute inflammatory response, ulceration, with the danger of intestinal perforation.
The onset of typhoid is insidious with headache being a prominent symptom. Other nonspecific symptoms include anorexia, malaise, general ache and pain, and fever. The fever is remittent and gradually increases in severity in a stepladder fashion over 3 to 4 days. Cough, sore throat, and altered behavior may be present. Constipation is usually present in the initial course of the illness.
During the second week, the fever becomes more sustained at around 104°F. At this stage the patient is severety ill with marked weakness and abdominal discomfort. Diarrhea becomes very prominent feature at this stage. An erythematous maculopapular rash that blanches on pressure appears on the upper abdomen and thorax. This rash, referred to as rose spot, lasts for only 2 to 3 days. Besides this rash several other physical signs can be elicited, such as splenomegaly , cervical lymphadenopathy, and hepatosplenomegaly. The majority of the patients have slight abdominal tenderness.
During the third week of illness, aptly referred to as ‘week of complications’, is the time when the majority of the complications occur. These include lobar pneumonia, hemolytic anemia, meningitis, polyneuropathy, acute cholecystitis, urinary tract infections, and osteomyelitis. Intestinal perforation and intestinal hemorrhage may occur in some patients. Fever continues in this week.
The fourth week of illness, referred to as ‘week of convalescence’, is characterized by a gradual return to health.
Typhoid carriers
Many patients of typhoid fever continue excretion of typhoid bacilli even after 1 month of recovery from the illness. Such persons are called convalescent carriers. There are, however, patients who continue excretion of typhoid bacilli for more than 1 year of recovery. They are referred to as chronic carriers. These carriers harbor typhoid bacilli in their biliary duct. Chronic carriers are of great epidemiological significance because they, being the reservoir of the infectious agent, play important role in the dissemination of the disease.
PARATYPHOID FEVER
Paratyphoid is caused by S. paratyphi A, and S. schottmuelleri. This illness is clinically indistinguishable from typhoid fever. However, it is of short duration and milder.
SALMONELLA BACTEREMIA
Two species of Salmonella, namely S. choleraesuis and S. typhimurium, are usually involved in bacteremia. This illness is produced as a consequence of presence of the salmonellae in the blood stream, transiently or for long periods of time. This condition may lead to metastatic infection, involving various body organs. Most common organs, which are involved, include bones, joints, heart, and meninges. Localization and abscess formation in these organs is the common clinical feature of bacteremia. The symptoms of bacteremia include fever and chills.
LABORATORY DIAGNOSIS
A suspected case of salmonellosis can be diagnosed either by bacteriologica method or by immunological method.
BACTERIOLOGICAL DIAGNOSIS
Diagnosis of saimoneiiosis depends on the isolation and identification of the suspected species from suitable clinical specimen.
Clinical specimens
Most suitable clinical specimens for the bacteriological diagnosis inciude fresh stool, urine, or blood. In typhoid or paratyphoid the suspected pathogen may be isolated from the blood during the first week or from the stool or urine in the second or third week of illness. in gastroenteritis, diagnosis rests on the isolation of the causative organism from stool.
Enrichment culture
In order to suppress the intestinal contaminants, the specimen is first inoculated in selenite F or tetrathionate broth. These media particularly promote the multiplication of S. typhi.
Isolation
After incubation for 1 to 2 days, the enrichment medium is inoculated on various media. The color differentiation makes it possible to select suspicious colonies and inoculate them as pure culture into other media necessary for their identification.
Identification
The isolated culture is identified on the basis of biochemical characteristics. For this purpose various biochemical test are performed.
IMMUNOLOGICAL DIAGNOSIS
The patients with typhoid and paratyphoid fever develop agglutinating antibodies to O and H antigens of S. typhi, S. paratyphiA, or S. schottmuelleri between the ‘ second and fourth weeks of iliness. Tests for these antibodies may be employed for more reiiabie serological diagnosis.
Clinical Specimen
Serum Sample should be obtained from the patient most preferably in the secbnd and third week of the illness. At least 2 specimens should be obtained at intervals of 7-10 days to prove rising titer.
Slide agglutination test
This is Simple and rapid qualitative method to demonstrate the presence of H and 0 antibodies in the patient’s serum. A drop of the serum specimen is mixed with known antigens on a glass slide. The mixture is observed under low power objective.Agglutinates if appear indicate a positive test.
Widal test
The immunological diagnosis of typhoid fever was first introduced by Widal; hence this test is referred to as the Widal test. For a definitive result serial dilutions of serum are tested by means of the standardized agglutination test with formalinized suspensions of S. typhi for the detection of H antibodies and heat-killed suspensions for the titration of 0 antibodies. Clumping of antigenic cells that is visible to the naked eyes indicates a positive test.
There is a definitive pattern of the appearance of H and 0 antibodies during the course of the disease. H antibodies appear late in the second week, and persist in high concentration for many years. 0 antibodies, on the other hand, appear early in the second week of the disease and persist for only a short period of time. 0 antibodies are shared by many different species of Salmonella and Gram-negative bacteria. Following immunization with typhoid vaccine antibodies against H antigen of S. typhi may be present in elevated titer for many years. For these reasons, both H and 0 antibodies are investigated to provide a more reliable diagnosis.
The procedure of Widal test involves the preparation of two-fold master dilutions of the given serum sample by serial dilution method. All the dilutions are prepared in saline. A standard quantity of each of the master dilutions is mixed with a fixed quantity of antigen in Widal tubes placed in a Widal rack. A control is also set in Widal tube in which saline is added in place of serum dilution. The Widal rack containing H antigen (H rack) is incubated at 37°C and the rack containing 0 antigen (0 rack) at room temperature. Observation for agglutination in H rack is made after 2 hours and in O rack after 18 hours.
Widal test may indicate a past or present infection. A present infection is indicated when a titer of 1:160 with 0 antigen is noted. This titer shows an increase in subsequent samples. A titer of 1:80 to 1:160 with H antigen only indicates a past infection, recent vaccination, or an anamnestic response. The presence of both 0 and H antibodies indicates a present disease.
THERAPY
Physicians usually recommend four antibiotics for the treatment of Salmonella
infections: ampicillin. amoxicillin, chloramphenicol, and trimethoprim_ sulfamethoxazole. All of these drugs are effective but increasing resistance is becoming widespread. This is why it is recommended to select a drug only after antibiotic susceptibility test. Ceftriaxone and Ciprofloxacin are also effective but they are not recommended for children and pregnant women. The treatment should be continued for two weeks. Complications such as perforation of ileum and hemorrhage may require surgery.
Treatment of carriers does not usually yield successful result though ciprofloxacin is recommended for four weeks. When chemotherapy fails, cholecystectomy proves the only remedy.
CONTROL AND PREVENTION
Salmonella infections can be controlled by taking the following measures.
* Isolation of patients. isolation of patients is not necessary but they should be kept in a separate room. The room should be screened from flies and
perfectly clean.
* Disinfection of excreta. The infectious excreta should be properly disinfected and disposed of.
* Disinfection of contaminated objects. Contaminated objects (such as, bedpans, bed linens, rectal thermometers, and clothing) should be adequately disinfected before their reuse. Disinfection techniques should be used frequently for toilet seats and other bathroom facilities.
* Complete bed rest. The patient should be advised to take complete bed rest.
He should not be allowed to handle food until stool and urine cultures appear negative.
* Practices in hospitals. In hospitals gowns should be worn and hand washing should be practiced.
* Medical facilities. Medical facilities should be made available to the patients. Treatment should continue until complete recovery from the illness is evident.
* Management of close contacts. Close contacts of the patients should not
be allowed to handle food. They may be actively immunized with vaccine.
Following measures may be recommended for the prevention of salmonella infections.
* Sanitary measures. Salmonella infections can be controlled by proper sanitation withh special emphasis on sewage disposal and maintenance of
unpolluted water supplies. Sanitary system should be designed in the light of scientific requirements.
* Food handling. Special attention is required to protect the processed food. Preparation of products by the food industries should be properly supervised. All the food handling personnel in the food industry plants, slaughterhouses, and restaurants should be trained and educated in hygiene practices.
* Storage of food. Another important control measure is adequate cooking and refrigeration of foods in food processing plants, restaurants, and homes. Special attention is required in the storage of the poultry and related products.
* Animal slaughtering. One of the useful control measures is altered and improved animal slaughtering practices to reduce cross-contamination of animal carcasses.
* Salmonellae-free animal feeds. The major reservoir of salmonellae is poultry and livestock. It is, therefore, significant to reduce the number of organisms harbored in these animals in order to reduce human exposure. In some countries, all animal feeds are treated to kill salmonellae before distribution. This results in marked reduction in the cases of salmonellosis.
* Detection of carriers. The asymptomatic carriers should be detected and properly treated. They should not be allowed to work particularly in food processing plants, hospital, and restaurants until they are properly treated. They should not be allowed to handle the food at home.
* Water purification. Adequate water purification of water is necessary to make it safe for human consumption.
* Personal hygiene. The patients, contacts, and carriers should be given special instruction in personal hygiene, in hand washing after use of toilet and before meals, and in the sanitation of the excreta when adequate public sewers are not available.
* Public education. The public must be educated as to the source and transmission of salmonella infections.
* Vaccination. The susceptible persons should be vaccinated against salmonella infections. Three types of vaccines are now available against typhoid fever. The parenteral monovalent typhoid vaccine is used, as it is less likely to produce local and systemic reactions. This vaccine provides a short-term protection, usually for one year. There is another vaccine that incorporates the Vi polysaccharide antigen gives protection for about three years. Another vaccine is prepared by using an attenuated strain of S. typhi (Ty 21a). This strain, developed by chemical mutagenesis, is deficient in certain enzymes and thus survives and proliferates for a short time when administered oraly. It develops a local immunity in the intestine. It is also very effective vaccine though it provides a comparatively short-term protection. Booster doses should be given after every one-year. No vaccines are available for nontyphoidal salmonellosis.
