Shigella and salmonella are not found

Информация об исследовании

ПЦР (полимеразно- цепная реакция) - это метод, который позволяет найти в клиническом исследуемом материале небольшой участок генетической информации (ДНК) любого организма среди огромного количества других участков и многократно размножить его.

Биологический материал: кал.
Правила подготовки: кал следует сдавать до начала приема антибиотиков и химиотерапевтических препаратов (если это невозможно, то не ранее чем через 12 часов после отмены препарата).

Энтероинвазивные E.coli.
Кишечная палочка(E.coli) - возбудитель эшерихиозов, основная аэробная часть микрофлоры кишечника. Это граммотрицательные палочковые бактерии, принадлежащие к семейству Enterobacteriaceae. E.coli является обычным обитателем кишечника многих млекопитающих и приматов, к числу котрых принадлежит человек. Поэтому её называют кишечной палочкой. В организме человека кишечная палочка выполняет полезную роль, подавляя рост вредных бактерий и синтезируя некоторые витамины. Однако существуют разновидности бактерий E.coli способных вызывать у человека острые кишечные заболевания. В настоящее время выделяют более 150 типов патогенных (энтеровирулентных) палочек E.coli, объеденённых в 4 класса:
-Энтеропатогенные (ЭПЭК)
-Энтеротоксигенные (ЭТЭК)
-Энтероинвазивные (ЭНЭК)
-Энтерогемморагические (ЭГЭГ)
Энтероинвазивные кишечные палочки - возбудители поражений весьма напоминающих бактериальную дизентерию.Патогенез тоже носит черты явного сходства: подобно шигеллам энтероинвазивные кишечные палочки проникают и размножаются в клетках эпителия кишечника. Как и шигеллы они неподвижны и не способны ферментировать лактозу.
Поражения характеризуются выраженными болями в животе и профузной водянистой диареей с примесью крови. На инвазивность указывает большое количество полиморфных ядерных лейкоцитов в испражнениях. Путь передачи: фекально-оральный.

Источник инфекции:
-Больной человек и животные
-Бактериносители
-Фекально-загрязнённые продукты питания и вода
Одним из подтверждающих методов диагностики эшерихиоза является ПЦР.
Биологический материал: кал.
Правила подготовки: кал сдается до начала приема антибиотиков и химиотерапевтических препаратов (если это невозможно, то не ранее чем через 12 часов после отмены препарата).

Сальмонелла (Salmonella spp.)
Сальманеллёзы - острые кишечные инфекции животных и человека, вызываемые сальмонеллами. Salmonella spp - это подвижные, грамотрицательные палочки, принадлежащие к роду Salmonella, семейства Enterobacteriacea (энтеробактерии).

Источник инфекции:
-Больные животные
-Больной человек
-Бактериносители

Путь передачи: алиментарный - через инфицированные пищевые продукты, как правило животного происхождения (мясо, мясные продукты, молоко, яйца, особенно утиные и гусиные, студень), при вынужденном неправильном убое животных, нарушений правил хранения и приготовления продуктов (соприкосновение готовой и сырой продукции, недостаточная термическая обработка продуктов перед употреблением и т.д.).

Клиническая картина.
Инкубационный период колеблется от 2-6 часов до 2-3 суток. Клинические проявления сальмонеллезов от бессимптомного носительства возбудителя инфекции до тяжелых септических форм.

Правила подготовки: кал следует сдавать до начала приема антибиотиков (если это невозможно, то не ранее чем через 12 часов после отмены препарата).

Кампилобактерии (Campylobacter spp.)
Кампилобактериоз - это острое инфекционное зоонозное заболевание, характеризующееся синдромом общей интоксикации, поражением желудочно-кишечного тракта и возможностью генерализации патологического процесса.
Кампилобактерии (Campylobacter spp.) представители семейства Campylobacteriaceae - мелкие необразующие спор грамотрицательные палочки.
В настоящее время в состав семейства Campylobacteriaceae входит три рода: Campylobacter, Helicobacter, Arcobacter.
Эпидемиология.

Клиническая картина.
Инккубационный перид: 1-10 дней (чаще 2-5 дней).

Salmonella and Shigella

Some Salmonella infections can bring dizziness to your elderly person. Dizziness is another plague of the elderly, keeping them from going shopping, getting to church and even from getting around their own homes. Drugs such as Antivert™ are given but only take the edge off the problem. Feeling dizzy can make your loved one home bound and stuck to a walker for every move.

Salmonellas, along with Shigellas, produce very toxic substances that cause dizziness. There are three common Salmonella varieties: Salmonella enteriditis, Salmonella paratyphi, and Salmonella typhimurium (386, 380, 354 KHz). Kill Salmonellas daily for a month by taking Lugol’s iodine (6 drops in a half cup water, after meals and bedtime, see Recipes). Unfortunately, this will not kill Shigellas; follow the Bowel Program (page 546) to get them.

During this time set up a system of sterilizing all dairy products (see Milk, page 425) since this is the source of reinfection. Set up a system of rinsing fingers (and fingernails) in 10% grain alcohol in the bathroom. Deli food and restaurant salads carry Salmonellas and Shigellas, too. Kill them, routinely, after eating such food due to necessity. A warm stomach full of food at a neutral pH is just the right culture condition for these bacteria. It’s like putting yeast into a bowl of warm water, flour and sugar. In half an hour it is overflowing with growth.

once Salmonella is entrenched in an organ it is difficult to eliminate. Only an electric zapper can kill them all (in an organ, not the bowel). If your body has the right conditions (like a low acid stomach) to let them grow you dare not swallow another one! Shigellas arrive with dairy foods, too, but prefer the lower intestine as their headquarters. Indigestion that starts right after eating is probably due to Salmonellas. If your indigestion comes in the night, this suggests Shigellas, since they’ve had time to reach their favorite place further down.

Campylobacter and E. coli, other digestive bacteria, are sometimes the culprits. The Bowel Program is effective against these also. Besides getting digestive improvement you get mental improvement, less depression, less dizziness, less irritability after clearing these up. Remember that eating bacteria and killing them later will not solve the problem. Stopping eating them will.

Digestion problems that remain after eliminating bacteria can be diagnosed in a rational way. Ask these questions:

• Is the stool orangish-yellow, or very pale, instead of greenish brown? If so, bile isn’t getting delivered to the small intestine from the liver.

• Is there abdominal pain? (More about this on page 97). It may be due to Ascaris, flukes, or other parasites.

• Is there constipation? This will let wastes accumulate, all the longer for bacteria to thrive on them.

• Is there bloating? This is due to gas made by bacteria.

• Does the stool float? If so, it must be lighter than water and contain fat or a great deal of undigested material.

Bile is necessary for digestion. Absorption of fat and calcium depends on bile mixing with the food. When fat isn’t absorbed, it stays in the intestine. Fat is lighter than water; it makes the stool float. Feces should not float. When the stool floats you can assume that calcium isn’t being absorbed either, leaving the blood in a deficit which will be taken from the bones.

If the stool floats or is orangish in color prepare your elderly person for a liver cleanse (page 552) to clear a bile duct of obstruction. They get quite fond of these cleanses and will ask to have one. Liver cleanses are completely safe, even for persons in their 80’s. One of the stones pictured on page 554 came from a woman age 97. The general rules apply to the extremely elderly: kill all parasites first by zapper if possible, otherwise by herbal parasite killing. Do a kidney cleanse (page 549) first, using half a dose instead of the regular dose, for three to six weeks. Attend your loved one in person for the liver cleanse, have a commode at bedside, protect bedding from accident: use paper underwear if necessary. share the joy of getting gallstones out painlessly with your loved one; let them see and count them if they wish before you flush them (use a flashlight).

Be extra careful with the skin cleansing. Hot water soothes and heals. Use starch skin soother to dispense onto the wet paper towel, besides borax solution and alcohol. Don’t use ordinary soap. The starch skin softener gives the smoothness of soap, and prevents the pain of friction. An elderly person may have no diarrhea at all with the Epsom salts! Evidently the body absorbs all the magnesium so eagerly, none is left in the intestine to absorb water and create diarrhea. It is especially important though to rehydrate your elderly person after a diarrhea. This time they do not balk at water consumption. The liver cleanse, it seems, gives them new thirst as well as new appetite. But it doesn’t last long. As the stones from the far corners of the liver move forward, they compact into larger stones and plug the ducts again. Their previous symptoms return. Try to give a cleanse once a month until the dark color of the stool returns and it no longer floats.

The benefits of a liver cleanse will last longer if valerian herb is taken the day after the cleanse and from then forward. It may be preventing spasm of the bile ducts. Use 2 oz. of the herb (cut) in 3 cups water. Simmer for 5-10 minutes, let settle or strain. Add honey to sweeten. Give a few tbs. every 4 hours (or 6 capsules) for several days followed by a daily dose at bedtime.

If constipation is a problem, use an herbal product rather than a drug until you have removed the cause. Cascara sagrada (half dosage) or prunes work for many people. Adding roughage to the diet is a good solution but often doesn’t work. If you try bran, you should add vitamin С and boil it, first, because it is very moldy. But even eating tree branches for supper won’t move a bowel that has the wrong bacteria in it.

Bacteria are part of the cause; and part of the result! Constipation increases the bacteria level which causes further constipation! You may solve the constipation problem immediately by zapping. Even though this kills some “good” with some “bad” bacteria, no harm is done. The stool is recolonized in one to two days.

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TERM Fall '10
PROFESSOR Dr.JoshNeufeld
TAGS Nutrition,Bacteria,Media, Staphylococcus aureus, mannitol, Serratia marcescens, Enterobacter aerogenes

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Salmonella-Shigella (SS) agar is a selective and differential medium . It is used for the isolation, cultivation and differentiation of gram-negative enteric microorganisms isolated from both clinical and non-clinical specimens such as from feces, urine, and suspected food items (fresh and canned foods). This medium is not recommended for the primary isolation of Shigella as some of Shigella strains may not grow on SS agar due to relatively high level of selectivity.

Other less inhibitory media used for the isolation, cultivation and differentiation of gram-negative enteric microorganisms are:

Desoxycholate Agar,
MacConkey Agar,
Eosin Methylene Blue (EMB) Agar,
Xylose Lysine Deoxycholate (XLD Agar), and
Hektoen Enteric Agar

Despite its name, Salmonella-Shigella (SS) agar is not suitable for isolating shigellae as it is inhibitory to most strains.

Composition of Salmonella-Shigella (SS) Agar and their function:

Lactose: fermentable carbohydrate
Beef extract, proteose peptone: provides the nitrogen, vitamins, and amino acids in SS Agar
Ferric citrate: Sodium Thiosulfate is also a sulfur source, and acts with Ferric Citrate as an indicator to detect hydrogen sulfide production.
Sodium thiosulphate &Sodium citrate: selective agents, providing an alkaline pH to inhibit Gram-positive organisms and suppress coliforms
Bile salts : The bile salts inhibit growth of gram-positive microorganisms
Brilliant Green/Neutral Red: pH indicator.
Agar: Solidifying agent.

Final pH: 7.0 +/- 0.2 at 25ºC

Principle

The presence of bile salts mixture and dyes (brilliant green) inhibits the growth of gram-positive species to a varying degree. Differentiation of enteric organisms is achieved by the incorporation of lactose in the medium. Organisms which ferment lactose produce acid which, in the presence of the neutral red indicator, results in the formation of red/pink colonies. Lactose non-fermenters form colorless colonies. The latter group contains the majority of the intestinal pathogens, including Salmonella and Shigella.

The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production as evidenced by colonies with black centers.

Preparation of the media

Suspend 60 g of the medium in one liter of deionized or distilled water.
Mix well.
Heat with frequent agitation and boil for one minute.
Sterilization in autoclave is not necessary.
Pour into plates
Let the agar solidify and store in the refrigerator (avoid freezing). Prepared culture media can be kept for at least a week in refrigeration.
Note: Various commercial suppliers now supplies ready to use culture plates.

Culturing the sample -->

Allow the plates to warm to room temperature and the agar surface to dry before inoculating.
Heavily inoculate and streak the specimen as soon as possible after collection.
If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface.
Streak for isolation with a sterile loop.
Incubate plates aerobically at 35-37°C 18-24 hours.
Examine colonial morphology.

Results

Lactose fermenter: If lactose fermentation occurs, the medium will turn red due to the acidic pH. e.g. Escherichia coli, Klebsiella pneumoniae gives red colonies.
Non-Lactose fermenter: Salmonella, Shigella, and other non-lactose fermenters appear as transparent or translucent colorless colonies. Colonies of Salmonella spp. may appear with or without black centers (depending on the species isolated).

Typical colonial morphology on Salmonella-Shigella Agar is as follows:

E.coli ……….……………………………..Slight growth, pink or red
Enterobacter/Klebsiella …..………..Slight growth, pink
Proteus .…………………………………..Colorless, usually with black center
Salmonella……………………………….Colorless, usually with black center
Shigella .…………………………………..Colorless
Pseudomonas .………………………….Irregular, slight growth
Gram-positive bacteria………………No growth

Precautions:

As SS Agar media contains components of animal origin (absence of transmissible pathogenic agents cannot be ruled out) so treat it as potentially infectious, and handle observing the usual universal blood precautions. Do not ingest, inhale, or allow the media to come into contact with skin.

References

Image source: Collin college

Presentation on theme: "SHIGELLAE The genus Shigella contains fewer species than the genus Salmonella and is antigenically less complex. Clinical dysentery may be caused by Shigella,"— Presentation transcript:

1 Diagnostic microbiology lecture: 4 Enterobacteriaceae Abed ElKader Elottol MSc. Microbiology 2010

2 SHIGELLAE The genus Shigella contains fewer species than the genus Salmonella and is antigenically less complex. Clinical dysentery may be caused by Shigella, Salmonella, Entamoeba histolytica, Proteus morganii, and viruses. Shigella dysenteria was isolated by Shiga in Japan in 1898.

3 SPECIES: 1. Shigella dysenteriae 2. Shigella flexneri 3. Shigella sonnei 4. Shigella boydii

4 CLASSIFICATION: 1. Non-mannitol-fermenters Shigella dysenteria 2. Mannitol-fermenters Shigella flexneri Shigella boydii Shigella sonnei

5 MORPHOLOGY AND STAINING:
- Short rods - Non-encapsulated - Non-motile - Non-spore former - Gram-negative

6 HABITAT AND TRANSMISSION
Shigella species are found only in the human intestinal tract. Carriers of pathogenic strains can excrete the organism up to two weeks after infection and occasionally for longer periods. Shigella are killed by drying. Shigella are transmitted by the fecal-oral rout. The highest incidence of Shigellosis occur in areas of poor sanitation and where water supplies are polluted.

7 CULTURAL CHARACTERISTICS
All members of Shigella are aerobic and facultative anaerobes. Grow readily in culture media at pH 6.4 to 7.8 at 10 oC - 40 oC, with optimum of 37 oC. After 24 hours incubation, Shigella colonies reaches a diameter of about 2 mm. The colonies are circular, convex, colorless, but moderately translucent with smooth surface, and entire edges. Small tangled hair-like projections can sometimes be seen at one or more points on the periphery of the colony. In XLD they appear pinkish to reddish colonies while in Heaktoen Enteric Agar (HEA), they give green to blue green colonies.

8 If a number of typical colonies present onto the original plate, a tentative diagnosis can be made by direct slide agglutination with polyvalent Shigella antiserum. In all instances, diagnosis should be confirmed by additional biochemical tests and by specific type agglutination. Biochemical Characteristics: =All ferment glucose, some ferments mannitol =They do not form acetyl-methylcarbinol, =Does not hydrolyze urea or liquefy gelatin =Citrate negative =TSIA (Alkaline slant over acid butt) =IMVIC V + - -

10 PATHOGENIC DETERMINANTS
O antigen: The ability to survive the passage through the host defenses may be due to O antigen. Invasiveness: Virulent shigella penetrate the mucosa and epithelial cells of the colon in an uneven manner. Intracellular multiplication leads to invasion of adjacent cells, inflammation and cell death. Cell death is probably due to cytotoxic properties of shiga toxin that interfere with protein synthesis. The cellular death and resulting phagocytosis response by the host accounts for the bloody discharge of mucus and pus and shallow ulcers characteristic of the disease. Other toxins: It has a protein toxin which may be neurotoxic, cytotoxic, and enterotoxic. The enterotoxic property is responsible for watery diarrhea.

11 PATHOGENICITY Shigella dysentery’s form a powerful exotoxin, it is associated with epidemics of bacillary dysentery. In man, shigellosis begins with symptoms of acute gastro-enteritis which is accompanied by abdominal pain and diarrhea. As it progresses, diarrhea becomes more frequent and is usually accompanied colicky pain. Later diarrhea losses its fecal characteristic and is followed by mucus with pus and blood. The disease is usually accompanied by fever and marked prostration. It is also known that children are more frequently attacked than adult persons and the symptoms are more severe.

13 LABORATORY DIAGNOSIS The only satisfactory method of laboratory diagnosis is to cultivate the bacilli from the patient. In the early stages of acute shigellosis, isolation of the causative organism from the feces is usually accomplished without difficulties by using the same special media and methods employed for salmonella

14 1. Cultivation of the bacilli from stool specimen during the first 4-5 days of the
disease. 2. Smears: Gram-negative bacilli appearing singly TSIA = Alkaline/acid (No gas no H2S) IMVIC reaction : V + - - 3. Serological examination with polyvalent and monovalent anti-sera.

18 Methods for identification of salmonella & shigella
1. Shigella are rarely encountered except in feces, Salmonella are also found in cultures of bile, blood, urine, abscesses and cerebrospinal fluid. 2. Microscopic examination of stained smears is of no use for the identification of the bacilli except the fact that it is a gram negative bacilli. 3. On general and selective media colonies are similar. On Blood agar they are smooth, gray and opaque. Most salmonella species produces H2S and therefore will be black on Bismuth Sulfite Agar.

19 Points of identifications between Salmonella and Shigella :
Feature Bacillary dysentery Typhoid fever Disease Non – motile Motile Motility Negative Positive H2S K / A K / A + TSIA reaction GIT Systemic Nature of infection Short period Lasting Immunity Endo and exotoxin Potent Endotoxin Metabolite

20 TREATMENT : 1. Water and electrolytes replacement.
2. Antibiotic therapy is required to eliminate the organism. Due to the emergence of resistant strains of shigella, antibiotic sensitivity, must be performed on any shigella isolate to determine suitable antibiotics: Sulfonamides, tetracycline, Chloramphenicol, ampicillin and streptomycin are known to be effective against shigella.

21 Immunity • Short lived; Preparation of oral live attenuated vaccine is on the way to stimulate mucosal IgA. Prevention • Sanitary precautions Good personal hygiene (hand washing).

23 SPECIES OF MEDICAL IMPORTANCE
1. Yersinia pestis Plague 2. Yersinia enterocolitica Yersinosis 3. Yersinia pseudotuberculosis Yersiniosis

24 General Characteristics
• Gram-negative rods (Coccobacilli) • Non-motile with the exception of Y.pseudotuberculosis and Y. enterocolitica (motile at 22oC) • Facultative anaerobe • Catalase positive • Oxidase negative

25 Yersinia pestis Plague is primarily a disease or rodents that could be transmitted to man. Plague occurs in three forms: BUBONIC, SEPTICEMIC and PNEUMONIC. Bubonic plague is the common form resulting from the cutaneous inoculation of Y. pestis by the bite of an infective flea. The incubation period is 2-7 days after exposure. The disease is characterized by sudden fever, shaking chills, headache and pain in the area of involved lymph nodes.

26 Untreated, bubonic plague can result in coma and death.
Classic bubonic plague entails an inflammatory response in the regional lymph nodes which, when enlarged, is referred to as bubo. Untreated bubonic plague may become septicemic plague. Virtually, 100% of the untreated septicemic plague infections are followed by pulmonary involvement and death. At this stage human to human transmission may occur. NB: Plague is an internationally reportable disease, and when suspected, must be reported to the local public health authorities.

27 The bubonic plague (also known as the Black Death), was killed more than 200 million people in the Middle Ages.

30 Septicemic plague Pneumonic plague

31 Collection of Specimen
aspirate : an aspirate from buboes is a good specimen if handled properly using a sterile syringe and needle and aseptic technique in collecting. Special precautions must be taken to avoid the spread of infection. Face masks, gloves and gowns must be routinly worn when in contact with the patient. Blood: Blood drawn aseptically is also good specimen for isolation of Y.pestis is septicemic patients. sputum: Early morning sputum obtained from deep couph, avoiding contamination with saliva. The patient must be instructed to rinse the oral cavity three to four times. Biopsy from infected lymph nodes.

32 Staining & Culture Media
Smears are stained with WAYSON to be examined for bipolar stained rods. Routine media, selective for gram negative enteric bacteria are good for isolation. BHIB, Trypticase Soy Broth, Blood Agar and Nutrient Agar are also used. Identification by Biochemical Tests: Yersinia pestis is differentiated from other Yersinia species by a negative urease negative test and carbohydrate fermentaion tests. Motility at 37oC and 22oC is also used.

33 Safety pin Bipolar stained rod

34 “fried egg” Non_hemolytic

35 Appears as small non-lactose fermenting colonies
MacConkey agar : Appears as small non-lactose fermenting colonies

36 Have a good growth Trypticase soy broth :
..-Incubate : 5% CO2, 35° C , 24 hrs Have a good growth

37 Serologic identification
1. Identification of suspected Y. pestis isolate: Commercially avialable antiplague serum is used to agglutinate cells. 2. Patient serum: (Detection of antibodies): Formalinized suspension of known Y. pestis cells is used as antigen. One drop of diluted antigen is added to one drop of patient`s serum. This mixture is shaken gently for minutes and examined for agglutination under low power magnification.

38 Antimicrobial Sensitivity testing
Routine sensitivity testing is unreliable because In-Vitro results commonly differ significantly from In-Vivo efficacy. For example, penicillin may show slight to wide zone of inhibition of Y. pestis, although penicillin has no In-Vivo activity against this organism. Tetracycline, Streptomycin or Chloramphenicol and sulfonamides are effective in treatment of plague.

39 Y. enterocolitica & Y. pseudotuberculosis
Both agents causes yersinosis which appears as enteric disorders, e.g., diarrhea, enteritis, terminal ileitis. Clinical symptoms can not be distinguished from those caused by other enteric pathogens such as salmonella and shigella. Faecal-oral rout is the common mode of transmission, although may be transmitted by bites of infective arthropods. Human to human transmission is well documented.

40 * Both of them can cause Yersinosis ..

41 Collection & processing of Specimen:
Stool, blood, excised mesenteric nodes, or appendices obtained during surgery. Yersinia Selective Agar: is an excellent selective medium for the isolation of Y. enterocolitica from stool or sputum. Cold Enrichment technique: Specimens that are grossly contaminated, are inoculated into buffered saline (pH ) and stored at 4-7 oC. Both organisms tolerate and even grow at these temperatures, other organisms gradually dies.

42 Biochemical Identification
Y. enterocolitica Growth at 4 oC and on NA/Mac. Agars Motile at 22 oC. Indole production= Variable Urease positive =Ornithine decarboxylase Positive Y. pseudotuberculosis The same as Y.enterocolitica and could be differentiated from it by a sorbitol negative test and negative ornithine decarboxylase.

43 Serology: Specific antigens prepared from each organism is useful in detecting the presence of specific antibodies in patient`s serum. Specific antibodies are used to agglutinate suspected cultures. Treatment: Streptomycin, Chloramphenicol, Tetracyclines.

44 - + +/- Lysine Ornithine Motility at RT (22-26°C) Urea Mannitol
Biochemical Characteristics of Yersinia species Reaction Yersinia species Y. pestis Y. pseudo- tuberculosis Y. enterocolitica Lysine - Ornithine + Motility at RT (22-26°C) Urea Mannitol Sorbitol +/- Voges- Proskauer Indole

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