Enteric bacteria(pseudomonas+salmonella) Dr.Asem shihabi. Jumanah Nayef Abu Asbeh

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1 15 Microbiology sheet #15 1. Gram-negative facultative anaerobic rapidly growing bacteria are divided into 2 major Lactose fermenter group which is represented by the Coliforms. 2. Lactose non-fermenter group which is represented by Proteus, Providencia, and Organella. These 2 groups of bacteria are found in the intestinal flora of humans and animals and are excreted by the feces and can contaminate food, water, etc. and return back to our intestines. They can produce infection if they reach a sterile part in the body such as the blood, meninges, wound, etc. Don't forget that within the group of lactose fermenters (coliform) are types that produce diarrhea, known as diarrhegenic E. coli which include the types: entero-pathogenic, enterotoxigenic, and entero-hemorrhagic. Enteric bacteria(pseudomonas+salmonella) Pseudomonas group They are Gram negative bacilli and can't be distinguished by gram stain from coliform bacteria group. It is a non-fermenter group because it causes less fermentation of sugars, especially Dr.Asem shihabi lactose and glucose. of humans and animals, usually in association with water and vegetation. WHY?: It is adapted to nature and manages to biodegrade large molecules which accumulate in the environment, thus, it can survive better in the environment than coliforms. Jumanah Nayef Abu Asbeh It can survive better in the hospital than coliform bacteria because the mechanism of the cell wall resists dryness, and it develops resistance against antimicrobial drugs and disinfectants more easily than coliform bacteria. Therefore, pseudomonas group is important in hospitals as a causative agent of Nosocomial infections, which are known as hospital-acquired infections. It is more resistant to environmental factors and disinfectants, for example, pseudomonas can survive in 70% alcohol whereas coliform can't. Also in chlorination of water, the pseudomonas can still survive in concentrations of chlorine of 2 to 3 parts of chlorine which can be expressed as 1-2 grams of chlorine per cubic meter of water. The pseudomonas group can survive in chlorinated water. Drinking water and tap water contain a few numbers of cells of pseudomonas but it will not harm us % of the population might carry pseudomonas aeruginosa or other pseudomonas in their intestines, while 100% of the population carries coliform. Thus, this group might be found in the intestines of some people, especially people who are treated with antimicrobial drugs, due to the selective pressures which support the growth of pseudomonas on the expense of other organisms. Pseudomonas aeruginosa

2 Microbiology sheet #15 Gram-negative facultative anaerobic rapidly growing bacteria are divided into 2 major groups: 3. Lactose fermenter group which is represented by the Coliforms 4. Lactose non-fermenter group which is represented by Proteus, Providencia, and Organella These 2 groups of bacteria are found in the intestinal flora of humans and animals and are excreted by the feces and can contaminate food, water, etc. and return back to our intestines. They can produce infection if they reach a sterile part in the body such as the blood, meninges, wound, etc. Don't forget that within the group of lactose fermenters (coliform) are types that produce diarrhea, known as diarrhegenic E. coli which include the types: entero-pathogenic, enterotoxigenic, and entero-hemorrhagic. Pseudomonas group They are Gram negative bacilli and can't be distinguished by gram stain from coliform bacteria group. It is a non-fermenter group because it causes less fermentation of sugars, especially lactose and glucose. It is widely distributed in the nature, but more in the environment than in intestinal tract of humans and animals, usually in association with water and vegetation. WHY?: It is adapted to nature and manages to biodegrade large molecules which accumulate in the environment, thus, it can survive better in the environment than coliforms. It can survive better in the hospital than coliform bacteria because the mechanism of the cell wall resists dryness, and it develops resistance against antimicrobial drugs and disinfectants more easily than coliform bacteria. Therefore, pseudomonas group is important in hospitals as a causative agent of Nosocomial infections, which are known as hospital-acquired infections. It is more resistant to environmental factors and disinfectants, for example, pseudomonas can survive in 70% alcohol whereas coliform can't. Also in chlorination of water, the pseudomonas can still survive in concentrations of chlorine of 2 to 3 parts of chlorine which can be expressed as 1-2 grams of chlorine per cubic meter of water. The pseudomonas group can survive in chlorinated water. Drinking water and tap water contain a few numbers of cells of pseudomonas but it will not harm us % of the population might carry pseudomonas aeruginosa or other pseudomonas in their intestines, while 100% of the population carries coliform. Thus, this group might be found in the intestines of some people, especially people who are treated with antimicrobial drugs, due to the selective pressures which support the growth of pseudomonas on the expense of other organisms.

3 Pseudomonas aeruginosa It is one of the most common and important species of pseudomonas and can be associated with any type of infection in the body including wound infection, lung infection, gastrointestinal infection, urinary tract infection, blood infection, meningitis, etc. Often hospitalized patients might acquire the infection: during operation, using invasive techniques, especially using urinary catheters, in which they introduce a plastic tube into the urinary tract to reach the urinary bladder and to relieve the patient from urine and so the patient doesn't need to move from his bed to the toilet, or in relation to the respiratory tract: introduction of intubation to help in the breathing process. These devices (urinary catheters and intubation) can be contaminated with Pseudomonas aeruginosa might survive on floors, bed sheets, instruments, and hands of doctors and nurses, so they can easily contaminate devices and produce infections. Especially in burn cases where the skin is damaged, usually all burn infections (the skin damage is of first degree, second degree, etc.) often acquire infection with pseudomonas aeruginosa because it is found in the environment and can easily contaminate the burn wound. When there is skin damage, the Pseudomonas aeruginosa manage to reside and produce infection, and the infection can be easily recognized as there is coloration of the site of infection which is usually blue and the formation of blue/green pus that has a fruity smell. Pseudomonas aeruginosa, in particular, produce an enzyme, which is an antibioticlike substance, called pyocyanin, and is a pigment released by Pseudomonas that has an effect similar to that of antibiotics, so it kills Staph. or Strept. and survives on the skin instead of Staph. and Strept., producing a more severe inflammatory reaction. Therefore, pseudomonas aeruginosa is a common cause of infection in hospitalized patients, especially following surgery in relation to urinary tract, respiratory tract, etc. Special feature of pseudomonas aeruginosa than other organisms: Often during treatment, it develops resistance against antimicrobial drugs more easily than other types of bacteria (compared to Escherichia coli or other organisms). In isolates of pseudomonas, you find them to be multiresistant, and this causes problems in treatment. Pseudomonas aeruginosa is very serious, produces serious infections, is more resistant to antibiotics and is more associated with complications and even death in comparison with other organisms.

4 Salmonella group It is lactose non-fermenter and is related to some extent to the coliform bacteria group. It is called salmonella in relation to a person called Salmon who has contributed for the science of this group. Like coliform bacteria, it is Gram-negative but is lactose and urease negative, and these features are important in the identification of salmonella. It can be motile due to the presence of large number of flagella which surround the cell body. It is usually motile and is rarely non-motile. In relation to infection in the body, it initiates three types of antibodies; especially when certain types of salmonella reach the bloodstream, they initiate the production of antibodies against: the H-antigen which is the flagella the O-antigen (somatic) which is the cell body, some types called salmonella typhi, which are the causative agent of typhoidal fever, carry thin layers that are like capsules (surface polysaccharides) which causes the production of antibodies against Vi (virulence) antigen. Due to widespread of salmonella in the nature, especially in animals (in poultry; chicken), it is found in the intestinal flora of poultry (like E. coli in our intestinal flora) and might cause problems, like diarrhea, or not. There are types of salmonella which are adapted to the intestines of animals, and if they reach our intestinal tract, they can't cause any disease. Generally, the disease caused by salmonella is called salmonellosis and is due to the presence of salmonella in our intestines or in relation of developing typhoidal fever, but it is preferred to be used in relation to gastrointestinal tract, i.e. when you say there is a case of salmonellosis, you mean there is a case of gastrointestinal salmonellosis or food poisoning salmonellosis. The Salmonella group is divided into 2 important causative agents of disease: gastrointestinal or food poisoning salmonellosis and typhoidal salmonella. Gastrointestinal or food poisoning salmonellosis In E.coli, we called it diarrhegenic and not gastrointestinal because the clinical features, whether entero-toxigenic, entero-pathogenic, or entero-hemorrhagic, are related to the colon and developing of diarrhea; the colon is usually involved, the small intestine is less involved, and the stomach is not involved. In relation to the gastrointestinal salmonellosis, the clinical features will be related not only to the intestines, but also to the stomach because once we have taken any food contaminated with salmonella such as "shawerma", mayonnaise, eggs,.. shells of eggs usually contain small numbers of salmonella. If large numbers of salmonella reach the intestines, we have to expect that we will develop gastrointestinal signs and symptoms.

5 Gastrointestinal signs and symptoms include: inflammation of the intestines causing FEVER, abdominal pain, and diarrhea; but before diarrhea, vomiting occurs because salmonella makes the stomach upset. In staph. aureus as a causative agent of food poisoning, the clinical features start by vomiting, and diarrhea might be associated with it or not; there is NO FEVER because there is no inflammation of the intestines but release of toxins, which is a feature of intoxication affecting the central nervous system, specially the vomiting center in the brain. Its incubation period, the time needed for the clinical features to be recognized, is 8-24 hours Initiation dose, the number of cells needed to cause infection, is large (millions of cells), and the large number of cells is found in food particles in which the cells multiply outside the body. Once it reaches the intestines, it continues to increase in number and starts to cause the infection Gastrointestinal salmonellosis is usually mild and self-limited in healthy people that are not suffering from immunocompromised conditions, not very young children and not in elderly and lasts only for hours Recovery usually occurs without the necessary use of antibiotics but might be dehydrated and need to replace the fluids by oral hydration by taking more fluids, and rarely requires admission to the hospital. It can be a serious disease for infants, elderly, and people suffering from immunocompromised conditions. The bacteria might penetrate the mesenteric lymphatic nodes, reach the blood stream, and produce septicemia or meningitis. At this point, it is necessary to treat the patient with antibiotic. In salmonella gastroenteritis, usually healthy carriers are present for a short period, where the organism is carried in the gallbladder and then is eliminated without any problems. Typhoidal salmonella Typhoidal salmonella is derived from typhoid; typhoid means high fever up to 42, associated with severe malaise (feeling of general discomfort or uneasiness), severe hallucinations, coma, and complications affecting to some extent the nervous system. Due to the high fever, the patient will be very weak and all his body is affected and might suffer from various complications. It is more dangerous and more associated with severe complications. There are only few serotypes which are S. typhi, and S. parathyphi A, B, and C. The serotypes are in relation to the composition of the cell wall and

6 have similar clinical features so you can't differentiate between S. typhi and paratyphi based on clinical features, and the only way to distinguish between them is in the laboratory. The infection might be initiated by few cells taken by contaminated water or by direct contact with an infected person. The initiation dose, number of cells needed to initiate the infection, is a small number, and they grow slowly. The small number starts to divide slowly in the intestinal mucosa, and penetrate the mesenteric lymph nodes and reach the bloodstream where it can reach any internal organ of the body (liver, spleen, gallbladder, etc.). The incubation period, that is the time for the clinical features to start to be recognized, is 1-3 weeks, and the infection process develops slowly. **The incubation period is often important for understanding the epidemiology of the disease. Often the organism can t reside only in the intestines; it penetrates the intestinal mucosa and is carried by the bloodstream, where it reaches the liver and spleen. The first important clinical feature in relation to Salmonella typhi or paratyphi infection is developing of hepatospleenomegaly (enlargement of the liver and spleen) because of the increase in the inflammatory reaction in them and can be easily recognized by clinical examination. Then it might cause diarrhea, constipation, high fever; the organism might reach any part of the body causing meningitis, urinary tract infection, and can cause necrosis and disease in any part of the body. If the patient didn't manage to suppress the multiplication of the organism and produce enough antibodies to control the infection, he will develop severe peritonitis; perforation of the intestines will occur and he will die due to complications in relation to liver abscesses, damage to peritoneal cavity, etc. Mortality in association with salmonella typhi and paratyphi might reach 30% of the infected persons, while with gastrointestinal salmonellosis, it is very rare for complications and death to occur. In our country, before the era of antimicrobial drugs and presence of the treatment of drinking water with chlorination, from 1930s-1940s till 1970s, typhoidal fever was a very serious disease in Jordan and has killed a lot of people. An outbreak usually occurs, involving thousands of people. For example in Al-Salt in 1976, three thousand people have been infected with typhoid from contaminated water. This disease was very important in the history of infectious agents. Now we are lucky to rarely see cases of typhoidal fever, but on the long run if there is a problem with drinking water or water treatment was not well controlled then typhoidal fever might appear again. During the natural infection of salmonella typhi or paratyphi, often we have a number of persons becoming healthy carriers, where they carry the organism in the gallbladder after their recovery, being more common in women than in men, 5 times greater in women than in men, because women are known to produce more gallbladder salts. salmonella typhi survive in the salts of gallbladder, leading later to inflammation of the gallbladder and an operation is needed to remove the gall bladder.

7 Lab diagnosis It is important to diagnose if an infection is caused by salmonella. We can't use MaConkey agar, like with E. coli and coliforms, which recognizes lactose fermenters. If we culture stool specimen in MaConkey agar, a large number of E.coli, coliforms, proteus, etc. will be present and we can't discover colonies of salmonella. Therefore, we have to use special culture media such as Salmonella-Shigella agar and Hektoen-Enteric agar. The pictures in slides: on the left: Hektoen-Enteric agar; on the right: Salmonella-Shigella agar). Hektoen-Enteric agar and Salmonella-Shigella agar: contain large amounts of bile salts, bile inhibits the growth of coliform bacteria and Gram-positive bacteria, in other words, it inhibits the growth of large number of intestinal facultative anaerobic bacteria and support the growth of salmonella. Thus, salmonella can be easily recovered from these culture media. Once salmonella has been recovered, salmonella typhi and gastrointestinal salmonella produce H 2 S producing black colonies on the culture media which can be easily recognized. Afterwards, we identify colonies by using certain biochemical tests to prove that it is salmonella or not and serotyping (no details are needed). In certain cases of typhoidal fever, the patient receives antibiotic treatment at home or antibiotic prescribed for one week by a doctor, but the antibiotic has not managed to eliminate the infection or control it. Now if there is no recovery and fever is still present after taking the antibiotic, the patient might seek advice of other physicians or get admitted to the hospital, especially if he has some complications such as bleeding in the intestinal tract, has more severe necrosis of the liver, hepatospleenomegaly, etc.; it is not easy to discover the organism by taking a sample from the intestines and culturing it. In relation to salmonella typhi and paratyphi, often the organism can be isolated from the stomach, urine, blood, meninges (if the patient develops meningitis), bone marrow in chronic cases, and many parts of the body. If the culture from stool and urine proves negative, then we might look for presence of specific antibodies against salmonella typhi by a test known as Widal test. Widal test This test measures the levels of antibodies against somatic O- and H-antigens of salmonella typhi and paratyphi, which is called titer or salmonella titer against typhi and paratyphi. Usually, in a clinical positive case infected with salmonella typhi or paratyphi, the titer must be above 160. If you take a second sample after one week, the titer will increase to 320, then 640 and so on (the titer will increase by 2-folds after each week).

8 If the titer is less than 160, then it is not a case of salmonellosis and is another case of other types of diseases that are misdiagnosed as typhoidal fever like Brucella/ Brucellosis/ Malta fever which might show clinical features similar to those of typhoidal fever. In general, using of culture or Widal test is enough to diagnose and start treatment with proper antibiotic. There is a vaccine available for salmonella but it is usually used in the army and not for civil population because typhoidal fever can be controlled by hygienic measurements, especially by controlling drinking water to be free of fecal contamination and to be chlorinated; the food is of less importance with typhoidal fever and is more important in gastrointestinal salmonellosis. "Success is the ability to go from one failure to another with no loss of enthusiasm" -Winston Churchill Done by: Jumanah Nayef Abu Asbeh