Salmonella - Microbiology Lecture Notes PDF

Salmonella Year: 2 Semester: 1 Module: GIT Prof. Rania Kishk Instructor information Name: Prof. Rania M. Kishk Department: Microbiology and Immunology department Office hours: Monday 11am-1pm  INTRO...

Salmonella Year: 2 Semester: 1 Module: GIT Prof. Rania Kishk Instructor information Name: Prof. Rania M. Kishk Department: Microbiology and Immunology department Office hours: Monday 11am-1pm  INTRODUCTION Salmonellae are often pathogenic for humans or animals when acquired by the oral route. They are transmitted from animals and animal products to humans, where they cause enteritis, systemic infection, and enteric fever. Prof. Rania Kishk Objectives: By the end of this lecture, students will be able to: 1.Discuss the general characteristics of Family Enterobacteriaceae. 2.Mention the pathogenic species of genus Salmonellae 3.Define the term food poising and Discuss Salmonella food poising 4.Describe the Salmonella typhi and paratyphi, as regards to: Morphologic and cultural characters Virulence and pathogenesis of disease they cause (Typhoid fever) laboratory diagnosis: Blood and stool culture, Widal test, diagnosis of carriers, Methods for prevention. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 1.Discuss the general characteristics of Family Enterobacteriaceae. According to their effect on lactose, they are divided into two groups: 1- Lactose fermenters (Coliforms)→ E.coli, Klebsiella, Citrobacter, Enterobacter. 2. Non-lactose fermenters → Salmonella, Shigella, Proteus. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 1- Discuss the general characteristics of Family Enterobacteriaceae. Antigenic structure Enterobacteriaceae have a complex antigenic structure. They are classified by more than 150 different heat-stable somatic O (lipopolysaccharide) antigens, more than 100 heat-labile K (capsular) antigens, and more than 50 H (flagellar) antigens. In Salmonella serotype Typhi, the capsular antigens are called Vi antigens. The antigenic classification of Enterobacteriaceae often indicates the presence of each specific antigen; for example, the antigenic formula of an E. coli may be O55:K5:H21. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 1- Discuss the general characteristics of Family Enterobacteriaceae. Antigenic structure 1- O antigens: The most external part of the cell wall lipopolysaccharide and consist of repeating units of polysaccharide. 2- K antigens: K antigens are external to O antigens on some but not all Enterobacteriaceae. Some are polysaccharides, including the K antigens of E. coli; others are proteins. K antigens may be associated with virulence. 3- H antigens: located on flagella Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 2- Mention the pathogenic species of genus Salmonellae The classification of salmonellae is complex The members of the genus Salmonella were originally classified on the basis of epidemiology; host range; biochemical reactions; and structures of the O, H, and Vi (when present) antigens. There are seven evolutionary groups. Nearly all the salmonella serotypes that infect humans are in group I which includes more than 1400 serotypes Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 2- Mention the pathogenic species of genus Salmonellae Currently, the genus Salmonella is divided into two species each with multiple subspecies and serotypes. The two species are: 1. Salmonella enterica Most human illness is caused by the 2. Salmonella bongori (formerly subspecies V). subspecies I strains S enterica contains six subspecies, which are written as S enterica subspecies enterica. 1. Subspecies I: S.enterica 2. Subspecies II: S.salamae 3. Subspecies IIIa: S.arizonae 4. Subspecies IIIb: S.diarizonae 5. Subspecies IV: S.houtenae 6. Subspecies VI: S.indica Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 2- Mention the pathogenic species of genus Salmonellae Furthermore, salmonellae can be classified by their serotype; according to somatic O antigens and flagellar H antigens. The 2 species of Salmonella and their respective subspecies consist of more than 2500 serotypes (serovars). Based on their serotype, Salmonella species (specifically S. enterica) are further classified as “typhoidal” and “nontyphoidal”. Genus: Salmonella Species: S. enterica Subspecies enterica (subspecies I) Serotype Typhi which can be shortened to S Typhi Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 2- Mention the pathogenic species of genus Salmonellae Typhoidal Salmonella refers to those specific serotypes that cause typhoid (“enteric”) fever, and include the serotypes Typhi, Paratyphi A, Paratyphi B, and Paratyphi C. Non-typhoidal Salmonella refers to all other serotypes. Four serotypes of salmonellae that cause enteric fever can be identified in the clinical laboratory by biochemical and serologic tests. 1. Salmonella Paratyphi A (serogroup A) 2. Salmonella Paratyphi B (serogroup B) 3. Salmonella Choleraesuis (serogroup C1) 4. Salmonella Typhi (serogroup D). Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 2- Mention the pathogenic species of genus Salmonellae Salmonella enterica is the most important species: The most medically important serotypes are Salmonella Typhi, Paratyphi A, B, C which cause enteric fever. Salmonella Typhimurium and Salmonella Enteritidis which cause salmonella food poisoning or enterocolitis. Salmonella Choleraesuis causes septicemia with metastatic abscesses Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 3- Define the term food poisoning and Discuss Salmonella food poisoning Definition: Food poisoning is an acute condition that affects a group of people sharing the same food. Manifestations: diarrhea and vomiting except botulism (affect nervous system) Common bacterial causes: – Staphylococcus aureus – Salmonella typhimurium and enteritidis – Clostridium botulinum – Clostridium perfringens – Bacillus cereus – Vibrio parahaemolyticus – Listeria monocytogenes Salmonella food poisoning is due to multiplication of organisms in the gut, so the incubation period is long (12-48hr) Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Objective 4 Describe the Salmonella typhi and paratyphi, as regards to: ▪ Morphologic and cultural characters ▪ Virulence and pathogenesis of disease they cause (Typhoid fever) ▪ laboratory diagnosis: Blood and stool culture, Widal test, diagnosis of carriers ▪ Methods for prevention. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Morphology and Identification Salmonellae is gram negative, non capsulated bacilli. Most isolates are motile with peritrichous flagella. Salmonellae is facultative anaerobes. They grow on simple media, but they almost never ferment lactose or sucrose (pale non-lactose fermenting colonies). They are indole negative and urease negative. They ferment glucose and maltose with production of acid only in case of S.typhi, acid and gas in case of S.Paratyphi They usually produce H2S. They survive freezing in water for long periods. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Pathogenesis and clinical findings S Typhi, S Choleraesuis, and perhaps Salmonella Paratyphi A and Salmonella Paratyphi B are primarily infective for humans The organisms almost always enter via the oral route, usually with contaminated food or drink. The mean infective dose to produce clinical or subclinical infection in humans is 10⁵– 10⁸ salmonellae. Salmonellae produce three main types of disease in humans, but mixed forms are frequent: 1. The “Enteric Fevers” (Typhoid Fever) 2. Bacteremia with Focal Lesions 3. Enterocolitis Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 A. The “Enteric Fevers” (Typhoid Fever) Enteric fever is a severe, systemic illness, caused by Salmonella Typhi (most common) or Salmonella Paratyphi A, B, C (endemic in Egypt). The source of infection is the stool or urine of cases or carriers. After ingestion of contaminated foods or beverages: – the salmonellae reach the small intestine – from which they pass through the epithelium via specialized M-cells overlying the Peyer’s patches – and then enter the intestinal lymphatics, multiplies in mesenteric lymph nodes with subsequent invasion into the bloodstream (bacteraemia) and persistent for 1 week (during the incubation period) – Via bloodstream the salmonellae are spread to many other organs (eg, liver, gall bladder, spleen, kidney and bone marrow). It multiplies in these organs then passes into the blood causing secondary bacteraemia (fever and signs of clinical illness appear) – The organisms also multiply within the intestinal lymphoid tissue and are excreted in stools. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 A. The “Enteric Fevers” (Typhoid Fever) From the gall bladder, the organism reinvades the intestine where it multiplies in Peyer’s patches and gut lymphoid tissues Incubation period : 10–14 days After the incubation period: fever, malaise, headache, bradycardia, and myalgia occur. The fever rises to a high plateau (39°C to 40°C) The spleen and liver become enlarged Rose spots, 1–4 mm blanching pink macules, are the classic cutaneous manifestation of enteric fever. Rose spots are typically observed on the chest and abdomen Abdominal symptoms may include diarrhea, constipation, and general abdominal pain; however, invasive diarrhea is typically not observed in typhoid fever. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 A. The “Enteric Fevers” (Typhoid Fever) Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 A. The “Enteric Fevers” (Typhoid Fever) Contrary to the elevated white blood cell counts (WBC) in patients with sepsis, the WBC inpatients with typhoid fever is normal or low. Complications of enteric fever: were intestinal hemorrhage and perforation, and the mortality rate was as high as 10–15%. With the advent of successful antibiotic treatment, the mortality rate has decreased to less than 1%. The principal lesions are hyperplasia and necrosis of lymphoid tissue (eg, Peyer’s patches); hepatitis; focal necrosis of the liver; and inflammation of the gallbladder, periosteum, lungs, and other organs have been also described. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 A. The “Enteric Fevers” (Typhoid Fever) Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 B. Bacteremia and Other Invasive Salmonella Infections Bacteremia and vascular infection occurs in approximately 8% of patients with non- typhoidal Salmonella infections (Salmonella Choleraesuis) (immunocompromised pt). Complications of Salmonella bacteremia: meningitis, septic arthritis, and osteomyelitis (Rare). Mortality associated with Salmonella bacteremia increases with duration of bacteremia and potential progression to septic shock; rates range from 14% to 60% in patients with concomitant endovascular invasion. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Enterocolitis This is the most common manifestation of Salmonella infection The mean duration for carriage of organisms Incubation periods: after resolution of the infection is 4–5 weeks; up to 7 days antibiotic therapy may increase the duration of carriage. Eight to 48 hours after ingestion of contaminated food or water, there is nausea, headache, vomiting, and profuse diarrhea Low-grade fever (38°C to 39°C) and abdominal cramping are very common clinical symptoms. Diarrhea is usually self-limited with a typical duration of 3 to 7 days. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Enterocolitis This is the most common manifestation of Salmonella infection Microscopic stool examinations typically show leukocytes, and red cells are less often observed. Blood culture results are usually negative stool cultures are positive for salmonellae Stool culture remain positive for several weeks after clinical recovery. Inflammatory lesions of the small and large intestine are present. Bacteremia is a rare (2–4%) complication, except in immunocompromised patients. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Pathogenesis and clinical findings Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Diagnostic Laboratory tests A. Specimens B. Bacteriologic Methods for Isolation of Salmonellae C. Serologic Methods D. Nucleic Acid Amplification Tests Diagnosis of enteric fever depends on the stage of the disease: 1. During the first week: – Isolation from blood 2. During second week: – Isolation from stool, urine – Serologic test Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Diagnostic Laboratory tests Specimen For the definitive diagnosis of enteric fever, Salmonella Typhi or Salmonella Paratyphi must be isolated in culture; appropriate specimens are: 1. blood 2. bone marrow 3. other sterile sites 4. Urine 5. intestinal secretions. Freshly passed stool is the preferred specimen for the diagnosis of non-typhoidal Salmonella Specimens collected during the early stages of the enteric illness have the highest yield for recovery of the causative organism. Collection of multiple stool specimens may enhance the recovery rate of Salmonella, as well as other enteric pathogens (eg, Shigella). Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Diagnostic Laboratory tests Specimen Blood cultures: are the most commonly used method of diagnosis Blood must often be taken repeatedly in order to increase the yield of recovering the organism. In enteric fevers and septicemias, blood culture results are often positive in the first week of the disease. The addition of stool cultures may increase the overall yield of recovering the causative organism. Bone marrow cultures: have the highest sensitivity (80% to 95%), they are clinically less practical for patients suspected to have enteric fever. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Diagnostic Laboratory tests Specimen Urine culture results may be positive after the second week of the illness. A positive culture of duodenal drainage establishes the presence of salmonellae in the biliary tract in patients who are carriers of the organism Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 B. Bacteriologic Methods for Isolation of Salmonellae 1. Differential medium cultures (Eosin methylene blue (EMB), MacConkey,) permits rapid detection of lactose non-fermenters (not only salmonellae and shigellae but also Proteus, Pseudomonas, etc). Gram-positive organisms are somewhat inhibited. Bismuth sulfite medium permits rapid detection of salmonellae, which form black colonies because of H2S production. Most salmonellae produce H2S. 2. Selective medium cultures: The specimen may also be plated on salmonella-shigella (SS) agar, Hektoen enteric (HE) agar, xylose-lysine desoxycholate (XLD) agar, or desoxycholate-citrate agar All of which favor growth of salmonellae and shigellae over other Enterobacteriaceae. Chromogenic agars specifically for salmonella recovery are also available. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 B. Bacteriologic Methods for Isolation of Salmonellae Salmonella species on TSI agar slant. On TSI agar slant, Salmonella spp. ferment glucose, but not lactose; they produce H2S and gas Salmonella species on HE agar. Salmonella spp. do not ferment the carbohydrates present in the HE agar; however, the organism produces H2S, and the ferric ammonium citrate in the HE agar result in the Salmonella colonies to appear black Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 B. Bacteriologic Methods for Isolation of Salmonellae 3. Enriched cultures The specimen (usually stool) can also be placed into selenite F or tetrathionate broth, both of which inhibit replication of normal intestinal bacteria and permit multiplication of salmonellae. After incubation for 1–2 days, an aliquot from this broth is plated on differential and selective media. 4. Final identification: Suspect colonies from solid media are identified by biochemical reaction patterns and slide agglutination tests with specific sera. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Serologic Methods 1- Slide agglutination test (Qualitative) Clumping, when it occurs, can be observed within a few minutes. This test is particularly useful for rapid preliminary identification of cultures. There are commercial kits available to agglutinate and serogroup salmonellae by their O antigens Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Serologic Methods 2- Tube dilution agglutination test (Widal test) (quantitative) Antibodies to Salmonella appear in the serum of patient during the second week of illness and reach maximum titre during the 4th week. Principle of widal test: Serial dilution of patient serum (1/20, 1/40, 1/80, 1/160….) are made in sets of test tube. To each set, a suspension of O and H antigens of the different salmonella is added. The tubes are incubated, then examined for the presence of agglutination.. The highest dilution of the serum which gives agglutination is the end titre. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Serologic Methods “Widal test” Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Serologic Methods (Widal test) Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 C. Serologic Methods (Widal test) For proper interpretation of Widal test, two serum samples separated by 10 days interval should be tested. The detection of rising titre in the second serum sample indicates active enteric fever. If the test is done during the 1st week, false negative result as the antibodies start to appear during the second week Non enteric infection by other salmonella or autoimmune diseases may cause non- specific rise of the antibody titre due to the presence of cross-reacting antibodies. In Egypt, titre below 1/80 are of no significant due to the endemicity of the disease in the area and previous subclinical infection. Early antibiotic treatment lowers the antibody titre due to reduction of the antigenic mass. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Interpretation of Widal test If only one serum sample is available, the following facts should be taken into consideration: High titre of O and H (≥1/160) or rising titre= active infection High titre of H only (≥1/160) or rising titre= past vaccination or past infection Alternative to Widal test include rapid colorimetric and EIA methods Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 D. Nucleic acid amplification test Several commercial NAATs are available for direct detection of salmonellae in fecal samples of patients with acute diarrhea. ASSIGNMENT: Q1- Mention the pathogenic species of genus Salmonellae Q2- Illustrate the Widal test and how to interpret? Q3- mention (in details) the bacteriologic Methods for Isolation of Salmonellae? Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Epidemiology The feces of persons who have unsuspected subclinical disease or carriers are a more important source of contamination than frank clinical cases that are promptly isolated, such as when carriers working as food handlers are “shedding” organisms. Many animals, including cattle and rodents are naturally infected with a variety of salmonellae and have the bacteria in their tissues (meat), excreta, or eggs. Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Epidemiology A- carrier After manifest or subclinical infection, some individuals continue to harbor salmonellae in their tissues for variable lengths of time (ie, convalescent carriers or healthy permanent carriers). They may carry the organism in the gall bladder and intermittently excrete in the stool. Three percent of survivors of typhoid become permanent carriers, harboring the organisms in the gallbladder; biliary tract; or, rarely, the intestine or urinary tract. B- Source of infection: Water, Milk and other dairy products (ice cream, cheese, custard)—Contamination with feces and inadequate pasteurization or improper handling Shellfish, Dried or frozen eggs, Meats and meat products (From infected animals or contamination with feces by rodents or humans) Household pets—Turtles, dogs, cats, exotic pets such as reptiles, and so on Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Prevention and control 1. Sanitary measures must be taken to prevent contamination of food and water by rodents or other animals that excrete salmonellae. 2. Infected poultry, meats, and eggs must be thoroughly cooked. 3. Carriers must not be allowed to work as food handlers and should observe strict hygienic precautions. 4. Vaccination (is recommended for travelers to endemic regions, especially if the traveler visits rural areas or small villages where food choices are limited) Prof. Rania Kishk Objective 1 Objective 2 Objective 3 Objective 4 Objective 5 Prevention and control Two typhoid vaccines are currently available in the United States: 1. Oral live, attenuated vaccine: Should not be given to children younger than 6 years of age. Contain attenuated strain of S.typhi Four oral capsules taken one every other day. The last dose should be given at least 1 week before travel to allow the vaccine time to work. A booster dose is needed every 5 years for people at risk. 2. Vi capsular polysaccharide vaccine for intramuscular use (0.5 ml) The vaccine contain the Vi capsular polysaccharides of S.Typhi. Should not be given to children