Diagnostic microbiologylecture: 3EnterobacteriaceaeAbed ElKaderElottolMSc. Microbiology
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Members of the Serratia genus were once known as harmless organisms that produced a characteristic red pigment.
Today, Serratia marcescens is considered a harmful human pathogen which has been known to cause urinary tract infections, wound infections, pneumonia and diarrhea.
Serratia bacteria also have many antibiotic resistance properties which may become important if the incidence of Serratia infections dramatically increases.
Serratia can be distinguished from other genera belonging to Enterobacteriaceae by its production of three special enzymes: DNase, lipase, and gelatinase.SPECIES:1. S. marcescens2. S. rubiddea3. S. liquefaciens
• Non lactose fermenter • Citrate: positive.
• V-P: positive • ODC: positive
• Lysine: positive • Indole: Negative
• TSI A/A: (NO gas) • DNase: positive
Specimen: Sputum, urine, and blood.
Culture:A red pigment is produced by colonies of Serratia on routine laboratory media which is more clear when the organism is grown at 22 “C in the dark (Prodiginines dye).
= Some strains are hemolytic.The picture was taken by an electron microscope of typicalSerratiacells.
1. Proteus mirabilis
2. Proteus penneri
3. Proteus vulgaris
4. Morganella morganii
5. Providencia alcalifaciens
6. Providencia stuartii
7. Providencia rettgeri.
On EMB, Endo and MacConkey Agar
The colonies usually exhibit swarming. Non-lactose fermenting (Colorless).Pathogenicity:
Proteus, like almost every other bacterium in this family, can cause urinary tract infections and hospital-acquired infections.
Proteus is unique, however, because it is highly motile and does not form regular colonies.
Instead, Proteus forms what are known as "swarming colonies" when plated on non-inhibitory media.
The most important member of this genus is considered to be P. mirabilis, a cause of wound and urinary tract infections.
Fortunately, most strains of P. mirabilis are sensitive to ampicillin and cephalosporin.
Unlike its relative, P. vulgaris is not sensitive to these antibiotics.
However, this organism is isolated less often in the laboratory and usually only targets immunosuppressed individuals.
P. mirabilis and P. vulgaris can be differentiated by an indole test for which only P. vulgaris tests positive.Diagnosis:
• Smear: Gram-negative bacilli
• TSIA K/AG+
• IMVIC V + - -
• Swarming and “Gun-powder-like odor”
• Strong urease positive.
• Phenylalanine Deaminase test = Positive
• Lysine –
• Hydrogen sulfide +
• MotileProteus strains are used in the Weil-Felix test for Rickettsial diseases:
1. Proteus OX K
2. Proteus OX 19
3. Proteus OX 2
Weil and Felix isolated strains of Proteus vulgaris from patients with typhus fever and found that the sera of the patients agglutinated particular Proteus strains designated OX19 and OX2.
The Proteus organism is not the cause of Typhus fever. The cross agglutination seems to be caused by the presence of an alkalistable polysaccharide which is also present in R. prowazeki. The agglutination of these particular strains by sera of patients withRickettsial infection is known as the “Weil-Felix reaction” .Morganella &ProvidenciaBoth are closely related to the Proteus group because both elaborate urease and produce indole.
They however, don`t produce hydrogen sulfide.
Morganella morganii is the only important species of this genus.
It can cause urinary tract and wound infections, as well as diarrhea.
Chloramphenicol is a good choice for treating Morganella infections.
• Ornithine +
• TSIA: A/A
• IMVIC reaction : + + - -
Virulence factors include: pectinases, cellulases, (which degrade plant cell walls), and also proteases, lipases, xylanases and nucleasesSALMONELLASALMONELLA
1. S. typhi (The typhoid bacilli)
2. S. paratyphi A (Produce paratyphoid fever)
3. S. paratyphi B (Produce paratyphoid fever)
4. S. paratyphi C (Bacteremia without intestinal involvement)
5. S. typhimurium (Food poisoning)
6. S. enteritidis (Food infection)
7. Salmonella cholerasuis (Hog cholera bacillus)
8. Salmonella pullorum (White diarrhea in children)
9. Salmonella gallinarum (Fowl typhoid bacillus(MORPHOLOGY AND STAINING:
• Short rods (bacilli)
• Motile with peritrichous flagella except S. pullorum and S. gallinarum.
• Gram-negativeHABITAT AND TRANSMISSION:
Most Salmonella species are found in the intestine of animals especially pigs, cows, goats, sheep, rodents, hens, ducks and other poultry.
S. typhi and S. paratyphi, however ,are usually found only in human., both of which are excreted in the feces and urine of infected patients.
Infection occurs via ingesting contaminated food or drinks.
How is it spread?
Contact with animals
Eating contamination food
Contact with patientsPATHOGENICITY:
Salmonella can cause any one of three types of Salmonellosis:
1. Acute gastroenteritis or food infection type.
2. Septicemia or acute sepsis similar to pyogenic infection.
3. Enteric fevers:
an acute infectious disease characterized by continuous fever, skin eruptions, bowel disturbances and profound toxemia.
Following entrance of the typhoid bacilli into the human body through the mouth there is always an incubation period of 7 to 14 days, before symptoms appear.
During this time, the organism penetrates the wall of the upper intestine and causes an inflammation.
Then reaches the blood via the lymphatic system were they circulate and may be localized in many internal organs especially in spleen, bone marrow, and gallbladder.
Serious complication of typhoid fever may be produced as a result of multiplication of the bacilli in organs other than the intestine.
The organism begin to disappear from the blood during the first week of the illness, and especially after the second week.
The disappearance of the organism from the blood is clearly associated with the development of specific antibodies.
NEPHROTYPHOID:This condition is an immune complex disorder of the kidneys and is characterized by fever, edema, marked albuminuria and hematuria.
OSTEOMYELITIS : Especially in children with sickle cell disease and thalassamia.
Typhoid in children can be found in the bone marrow. Inflammation of the joints (Typhoid arthritis) may also occur.
ABSCESSES: of the spleen and elsewhere.
MENINGITIS And rarely pneumonia and endocarditis.
1. Surface antigens: The ability of Salmonella to attach to host receptor cells and to survive intracellularly may be due to the O antigenic side chain or in case of typhi serotypes, the presence of Vi antigen. Organisms containing Vi antigen are clearly more virulent than those lacking this antigen. It may function as a capsule and prevent phagocytosis or intracellular destruction of the organism.
2. Invasiveness: Virulent Salmonella penetrate the epithelial lining of the small bowel, thus the brush border begins to degenerate. After penetration the organism multiply and may spread to other body sites.
3. Endotoxins: Presumably, endotoxin is responsible for the fever production.
Endotoxin activation of the chemotactic properties of the complement system may cause the localization of leukocytes in the classic lesions seen in typhoid fever.
4. Other factors:
Enterotoxin: Affects the small intestine.
Cytotoxin: Associated with outer bacterial membrane which may mean that the toxin may be important in cellular invasion and cellular destruction.
The Kauffman-White system used to classify Salmonella is based on identifying the O (somatic) and H (flagellar) antigens possessed by the different serovars.
The detection of Vi antigen is also used in the identification of S. typhi and some other Salmonella.
Salmonella are grouped by their O antigens as groups A to Z, 51-61 and 64-66. Many of the medically important salmonella belongs to groups A to G.
Each group has what is called a group factor. This is an O antigen, common to all members of the groups. For example, the factor for group B is O antigen is 4.
This means that all the salmonella belonging to this group possess antigen 4 as one of their antigen.
2. H Antigen: Many Salmonella are diphasic, that is, they can occur in two antigenic forms referred as phase I and Phase II. Phase I antigens are given alphabetical letters and phase II antigens either numbered or given a letter if known to occur in both phases.
Phase I antigens are more specific and therefore, an organism can be identified if it is in phase I.
3. Vi Antigen: This surface (K) antigen can be found in S. typhi and S. paratyphi C and few other Salmonella. It is associated with virulence and can be detected using Vi antiserum.
SPECIMEN: For the diagnosis of enteric fever, specimens include, blood, feces and urine for culture may be used depending on the course of illness.
A. Blood: Organisms can usually be detected in 75-90% of patients during the first 10 days of infection and in about 30% of patients during the 3rd. week.
B. Feces: Organisms can usually be isolated from 40-50% of patients during the third week.
C. Urine: Organism, can usually be detected from about 25% of patients after the second week of infection.
D. Serum: Is used for the detection of serum antibodies (Widal test).
Due to the high cost of antisera, it will not be possible for most laboratories to stock wide range of Salmonella antisera, however, laboratories try to stock a salmonella polyvalent O antiserum which covers the locally important group and also specific O, H, and Vi antisera to identify S. typhi.
A. ENRICHMENT & SELECTIVE MEDIA:
Various enrichment and selective media are used to isolate Salmonella from stool and other specimens. The use of Selinite-F and XLD, DCA and SSA.
Salmonella produce non-lactose fermenting colonies on MacConkey medium. Most strains (especially food-poisoning serovars) show a blackening of the colonies due to H2S production.
Contains substances which have a growth stimulating of Salmonella and Shigella and inhibitory to the other contaminants. Ex. Selenite-F Broth Tetrathionate broth.
Solid media containing lactose as differential sugar; an indicator to produce color changes when the pH of the colony becomes acid as a result of lactose fermentation and an inhibitor for gram positive bacteria and most gram-negative bacteria other than Salmonella and Shigella.
1. Bismuth sulfide agar (BSA) : Considered by many as the best medium for the isolation of Salmonella typhi. Develops black colonies.
2. Brilliant Green agar (BGA): This medium is good for isolating Salmonella species other than S. typhi. Slightly pink colonies with black center
3. Salmonella Shigella agar (SSA): Colorless colonies with black center .
4. Desoxycholate Citrate agar (DCA) :Slightly pink colonies with black center .
5. Xylose lysine dextrose agar (XLD): Develops black colonies.
Desoxycholate Citrate agar (DCA)
Salmonella Shigella agar (SSA):
A. TSIA: Alkaline slant, Acid butt with small amount of H2S
B. IMVIC reaction: - + - +
C. Motility (+)
D. Urease (-)
D. SEROLOGICAL TESTING:
1. On clinical isolates: All clinical suspected Salmonella isolate must be subjected to serotyping by polyvalent and monovalent antisera.
2. On patients: Serological test (Widal test) must be performed after 10 days of infection .
• Antimicrobials not indicated
• Chloramphenicol was firstly used in 1948 for typhoid fever - Now resistant
• Ampicillin, sulphamethoxazole-trimethoprim still used in developing countries.
• Ciprofloxacin – drug of choice
• Rehydration if needed
• Prolonged treatment with Ampicillin or Ciprofloxacin
• Cholecystectomy may be needed
• Public health = personal hygiene measures
• Proper sewage treatment
• Chlorination of water supplies
• Detection of carriers
• Pasteurization of milk and proper cooking of food
Vaccination (50-80%) protection) – two vaccines:
• Acetone-killed – given I/M
• Live-attenuated - Oral
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END OF LECTURE
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