İnsan bağırsağında yaşayan Shigella basilleri, maymunlar dışında sadece insanlarda hastalık meydana getirir. Shigella cinsi içinde Shigella dysenteriae (en ağır seyirli olan), Shigella flexneri, Shigella boydii, Shigella sonnei olmak üzere 4 tür bakteri bulunur.


Morfolojisi

Shigella hareketsiz, sporsuz, kapsülsüz, gram negatif (-) basildir. Hareketsiz olmaları ile Salmonellalardan ayrılır; bakteriyolojik boyalarla kolay boyanır, Shigella flexneri'nin bazı tipleri dışında, Shigella basillerinde fimbria yoktur.


Üreme Özellikleri

Shigella, aerob veya fakültatif anaerob bakteridir. Optimal üreme ısısı 37oC‟dir; ancak 8-40 oC‟de de üreyebilir. Optimal pH‟sı 7.2-7.4‟dür. Gaita içinde bulunan koliform bakteriler tarafından hızla asitleştirildiği için birkaç saatte ölür. Dış ortam koşullarında oldukça dirençlidir. Isıya, güneş ışığına ve antiseptiklere dirençli değildir. Klorlu sularda harap olur. Adi besiyerinde kolay ürer, buyyonda homojen bulanıklık yapar. Jelozde yuvarlak, hafif kabarık, düzgün yüzeyli, saydam, E.coli kolonilerine benzer S tipi koloni oluşturur. Shigella sonnei kolonileri, diğerlerine göre daha büyüktür. Endo, EMB, MacConkey besiyerlerinde renksiz koloni oluşturur. S.sonnei hariç, laktoz negatiftir (-).TSİ besiyerinde asit yapar, gaz oluşturmaz, H2S yapmaz, üre ve sitrat negatiftir. Shigella dysenteriae hariç, diğer Shigellalar mannit (+) tir.
                                       

Morphology   
➧Small Gram Negative (-), facultatively anaerobic, coliform bacillus
 ➧Non-motile (no H antigen)
 ➧Possess capsule (K antigen) and O antigen
  ➤ K antigen not useful in serologic typing, but can interfere with O antigen determination
  ➤ O antigens: A, B, C, D correspond respectively to the four species
➧Non-lactose fermenting
➧Bile salts resistant: trait useful for selective media.



➤ Family Enterobacteriaceae
➤ All Enterobacteriaceae
➧ferment glucose
➧reduce nitrates (NO3 to NO2 or all the way to N2)
➧are oxidase negative


 Four species:
➧Shigella dysenteriae: causes most serious form of bacillary dysentery
➧Shigella flexneri: most common cause of shigellosis in underdeveloped countries
➧Shigella sonnei: most common cause of shigellosis in developed countries
➧Shigella boydii


Clinical Syndromes (Shigellosis):
  Ranges from asymptomatic infection to severe bacillary dysentery.
  Two-stage disease: watery diarrhea changing to dysentery with frequent small stools with blood and mucus, tenesmus, cramps, fever
Early stage:
➧Watery diarrhea attributed to the enterotoxic activity of Shiga toxin
➧Fever attributed to neurotoxic activity of toxin
➧Process involves:
1-Ingestion
2-Noninvasive colonization and cell multiplication
3-Production of the enterotoxin by the pathogenic bacteria in the small intestine;


  Second stage:
➤ Adherence to and tissue invasion of large intestine.
➤ Typical symptoms of dysentery.
➤ Cytotoxic activity of Shiga toxin increases severity.


Epidemiology:
➧ Shigellosis is a major cause of diarrheal disease throughout the developing nations of the world
➧ Major cause of bacillary dysentery (severe second stage form of shigellosis) in pediatric age group (1-10 years old) via fecal-oral transmission
➧ Estimated 15%-20% of pediatric diarrhea in United States
➧ Leading cause of infant diarrhea and mortality (death) in developing countries
➧ Shigella occurs naturally in higher primates, so shigellosis is spread from human to human via the fecal-oral route primarily by contaminated hands (ingestion is portal of entry)
➧ Less frequently, transmission by ingestion of contaminated food or water
➧ Outbreaks usually occur in close communities; Daycare facilities account for a large proportion of cases that occur in the U.S.
➧ Secondary transmission occurs frequently
➧ Low infectious dose (102-104 CFU) with 1-3 day incubation period
➧ Carriage of the organism persists for approximately one month following convalescence



Laboratory Identification:
➧ Closely related to Escherichia
➧ Four distinct species differentiated on basis of serogrouping and biochemical analysis
➧ Stool specimens and rectal swabs should be cultured soon after collection or placed in appropriate transport medium (e.g., Cary-Blair medium)
➧ Readily isolated on selective/differential agar media (e.g., XLD, SS, and brilliant green agar
➧ Lactose nonfermenter


Treatment, Prevention & Control:
➤Dehydration is problem to attend
➤Treat carriers, major source of organisms; Ampicillin and trimethoprim-sulfamethoxazole are effective antibiotics that reduce duration of carriage
➤Antibiotic resistance is a major problem
➤Proper sewage disposal and water chlorination
➤Oral vaccines of Shigella: E.coli hybrids or Shigella mutants offers immunity for six months to one year.




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The genus Klebsiella consists of non-motile, aerobic and facultatively anaerobic, Gram negative rods. At the time of writing, the genus Klebsiella comprises K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp.ozaenae, K. pneumoniae subsp. rhinoscleromatis, K. oxytoca, K. ornithinolytica, K. planticola, and K. terrigena. However, comparison of the sequences of each species shows that the genus is heterogeneous, and may be more reasonably arranged in three clusters. Cluster 1 contains the three subspecies of  K. pneumoniae, cluster 3 contains K. oxytoca and cluster 2 contains the other species (which are notable for growth at 10° C and utilization of L-sorbose as a carbon source). It has been proposed that the genus Klebsiella be divided into two genera and one genogroup, with the name Raoultella being the genus name for those organisms in cluster 2.

For the purpose of conforming to current clinical usage, the clinically important species and subspecies will be referred to as K. pneumoniae, K. ozaenae, K. rhinoscleromatis and K. oxytoca in this chapter.

LABORATORY DIAGNOSIS

The members of the genus Klebsiella are Gram negative, nonmotile, facultative anaerobic rods ranging from 0.3 to 1.0 μm in width to 0.6-6.0 μm in length. Most strains grown readily on standard media, although occasionally cysteine requiring urinary isolates of K. pneumoniae are encountered. These strains will appear as pinpoint colonies on routine media, and require supplementation of media with cysteine for more adequate growth. The vast majority of Klebsiella spp. are encapsulated - contrary to popular belief it is probably not capsule which primarily contributes to the mucoid appearance that some Klebsiella strains exhibit.  The Klebsiella which has been linked to the invasive syndrome presenting as liver abscess have a mucoid appearance.

In practice, K. pneumoniae and K. oxytoca are distinguished by indole production by K. oxytoca but not K. pneumoniae. It should be noted however that K. ornitholytica is also an indole producer. The five clinically important species can be distinguished by tests for indole production, ornithine decarboxylase production, he Voges-Proskauer reaction, malonate utilization and o-nitrophenyl-β-D-galactopyranoside (ONPG) production.


Production of plasmid-mediated extended-spectrum beta-lactamases (ESBLs) by Klebsiella spp. has become a major problem. The nature and characteristics of ESBLs are described in greater detail below. Detection of ESBLs in clinical isolates of Klebsiella spp. is problematic since a significant proportion of ESBL producing isolates appear susceptible to third generation cephalosporins or aztreonam. Yet, poor clinical outcomes have been observed when these same antibiotics have been used to treat serious infections due to apparently susceptible ESBL producers. A single surrogate marker for ESBL production, such as ceftazidime resistance, is insufficient for the detection of ESBLs. Virtually all reliable laboratory tests used for detection of ESBLs rely on the change in in vitro activity of oxyimino containing beta-lactams in the presence of a beta-lactamase inhibitor such as clavulanic acid. Examples of ESBL detection methods include the double disk diffusion test, Etest strips containing ceftazidime or cefotaxime with and without clavulanic acid, the Vitek ESBL detection card and the Microscan ESBL plus detection system. Clinical and Laboratory Standards Institute (CLSI) has also developed screening and confirmatory tests for detection of ESBLs. It should be noted that these are standardized for K. pneumoniae and K. oxytoca only.


In some circumstances there is a need to detect ESBL producing Klebsiella spp. from stool or rectal swabs. Examples of such media include Drigalski agar supplemented with cefotaxime 0.5 mg/L , MacConkey agar supplemented with ceftazimide 4 mg/L and nutrient agar supplemented with ceftazimide 2 mg/L, vancomycin 5 mg/L and amphotericin B 1667 mg/L.

1. Touch a well isolated colony with a sterile straight wire.
2. Inoculate TSI by first stabbing through the centre of the medium to the bottom of the tube and then streak the surface of the slant.
3. Leave the cap loose and incubate the tube at 35 in ambient air for 18 to 24 hours.
4. Observe the reaction

Glikoz, laktoz ve sükroz olarak bakılır. Yatık TSI besiyerine inoküle edilen suş 37°C’ da 24 saat inkübe edilir. İnkübasyon sonunda tüpün dibindeki sarıya dönen renk değişimi glikozun kullanıldığını, yatık yüzeydeki sarıya dönen renk değişimi laktozun kullanıldığını, yatık yüzeyin ucundaki sarıya dönen renk değişimi sukrozun kullanıldığını gösterir. Ayrıca (Norveç üçlü tüp metodunda kullanılan ikinci besiyerine) mannitol’ e geçilerek 37°C’ da 24 saat inkübasyondan sonra sarıya dönen renk değişimi ile mannitol fermantasyonu saptanır.

Objective

To determine the ability of the organism to hydrolyse urea by the action of urease enzyme.

Principle

Urea is a nitrogen-containing compound that is produced during decarboxylation of the amino acid arginine in the urea cycle. Urea is a major organic waste product of protein digestion in most vertebrate and is excreted in urine. Some bacteria have the ability to produce an enzyme urease as part of its metabolism to break down urea. The urease is a hydrolytic enzyme which attacks the carbon and nitrogen bond with the liberation of ammonia and carbon dioxide. It is a useful diagnostic test for identifying bacteria, especially to distinguish members of the genus Proteus from Gram-negative pathogens. Proteus vulgaris is an important and fast producer of urease.

Urease test is performed by growing test organism on urea agar slant or urea broth with phenol red as an indicator with pH6.8. During the incubation period, the organism capable of producing urease enzyme hydrolyse urea and produce ammonia that raises the pH level. As the pH increases, the phenol red changes from yellowish orange as the initial colour of medium to deep pink. Failure of development of a pink colouration due to no ammonia production is evidence of an inability of the organism to produce urease enzyme.

Media

Enzymatic digest of gelatin (1 g), dextrose (1 g), NaCl (5 g), KH2PO4 (2 g), urea (20 g), phenol red (0.012 g), per 1000 mL, pH 6.8.

Procedure

1. Streak the surface of a urea agar slant with a portion of a well-isolated colony or inoculate test organism on urea broth containing phenol red as the indicator.
2. Incubate the urea agar slant or urea broth at 37°C for 24-48 hours.
3. Examine the development of pink colour.
4. In case of unknown result incubate the tube for 7 days to check for slow urease production.

                               

1. Proteus spp
2. Corynebacterium spp
3. Helicobacter pylori
4. Brucella spp

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Methyl Red Test (MRVP broth) 

Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.

 Description: Mixed acid fermentation -Many gram-negative intestinal bacteria can be     differentiated based on the products produced when they ferment the glucose in MR-VP medium. Escherichia, Salmonella, andProteusferment glucose to produce lactic, acetic, succinic, and formic acids and CO2, H2, and ethanol. The large amounts of acids produced lowers the pH of the medium -Methyl red (a pH indicator) will turn red when added to the medium if the organism was a mixed acid fermenter. Many of these organisms also produce gas.

Interpretation: After incubation: The broth must be turbid. A clear broth indicates that your organism did not grow and cannot be tested. Remove 1 ml of broth and place into a sterile tube before performing the methyl red test ifyou are going to use the same broth for the VP test. Add 3-4 drops of methyl red to the original broth. DO NOT SHAKE THE TUBE. A positive result has a distinct red layer at the top of the broth. A negative result has a yellow layer.


Voges-Proskauer Test

Procedure: Inoculate a loopful of bacteria into MRVP broth. Incubate 3 to 5 days.

Description: Organisms that are negative in the methyl red test may be producing 2, 3 butanediol and ethanol instead of acids. These non-acid products do not lower the pH as much as acids do.Enterobacter, Serratiaand some species of Bacillus produce these substances. There is no satisfactory test for determining production of 2, 3 butanediol. A precursor of 2,3 butanediol called acetoin can be detected with Barritt's reagent.

Interpretation: After incubation: Read the VP test when you have good turbidity. A clear broth indicates that your organism did not grow and cannot be tested. Barritt's reagent A (VP A) contains naphthol and Barritt's B (VP B) contains KOH. Test 1 ml of your culture from the MRVP broth. If you have already conducted the methyl red test, you should have already placed 1 ml of untested broth in a sterile tube. If you haven’t done this, do so now. Add the entire contents of the VP A reagent (15 drops) and 5 drops of the VP B reagent to the 1 ml of your broth culture. SHAKE WELL. This reaction will take a few minutes before you will see a color change. SHAKE the tube every few minutes for best results. With a positive reaction the medium will change to pink or red indicating that acetoin is present. With a negative reaction the broth will not change color or will be copper colored. Wait at least 15 minutes for color to develop before calling the test negative.





Citrate Test (Simmon's Citrate Slant)

Procedure: Streak a loopful of bacteria onto a citrate agar slant, do notstab the butt. Incubate 24 to 48 hours, longer forBacillus species. Incubate with a loose cap.

Description: Simmon's citrate agar tests for the ability of an organism to use citrate as its sole source of carbon. This media contains a pH indicator called bromthymol blue. The agar media changes from green to blue at an alkaline pH.

Interpretation: After incubation: A positive reaction is indicated by a slant with a Prussian blue color. A negative slant will have no growth of bacteria and will remain green.