1. Fastidious Gram Negative Rods Respiratory Culture Unit
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Fastidious Gram Negative Rods Respiratory Culture Unit
Hi I’m Carol Larson, your guide thru this tutorial about the fastidious gram-negative bacilli. This lesson will be of value to you as you rotate thru your Respiratory Culture Rotations and your Blood Culture Rotation. You will find an audio icon on each screen of this presentation. Please click on the icon to hear my narration for that specific screen. You may also follow along with the lecture handout entitled “Fastidious Gram Negative Rods” that you can print out from this blackboard lesson.
Besides Haemophilus species that we discussed in student lab, there are many other fastidious gram negative rods. As you will recall, the second page of objectives in your Haemophilus section of student lab notes related to these other fastidious GNRs. This supplemental lesson will help to bring you up to speed on key characteristics of the fastidious gram-negative rods.
Division of Medical Technology
Carol Larson MSEd, MT(ASCP)

2. General Information Fastidious Faint staining Gram Negative Rods
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General Information
Fastidious
Complex / extensive nutrient requirements
Faint staining Gram Negative Rods
Safranin counterstain for >2 minutes
Substitute carbolfuschin for safranin
Serological testing useful
Fastidious is defined as an organism having complex or extensive nutritional requirements. These organisms generally will not grow on the general purpose culture media that we use in the lab.
These organisms appear as faintly staining gram negative rods. It is beneficial to try and enhance the gram reaction by two methods:
We can extend the time for the safranin counterstain to at least 2 minutes, or we can substitute carbolfuschin for the safranin stain.
In addition, serologic test methods are useful in the direct detection, identification and diagnosis of these pathogens. These methods can include: fluorescent antibody staining, direct agglutination testing, DNA probes, and enzyme immunoassay.

3. Bordetella pertussis Clinical Significance
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Bordetella pertussis Clinical Significance
Causes Pertussis / Whooping cough
Spread by airborne droplets
Virulence factors
Attachment to ciliated epithelium of the upper respiratory tract
Exotoxin – tracheal cytotoxin
Exotoxin - Pertussis toxin
Cell wall endotoxin
Bordetella pertussis causes Whooping Cough or Pertussis in children. It is strictly a human pathogen. It is transmitted from person to person by airborne droplets from the cough of an infected person.
Bordetella pertussis has several virulence factors that aid it in causing infection. It first adheres to the ciliated epithelial cells of the upper respiratory tract. It then produces a cytotoxin that paralyzes the cilia. The major virulence factor is the pertussis toxin which it releases into the bloodstream. The PT is able to disrupt the patient’s immune system. A cell wall endotoxin is able to block the access of the host’s lysozyme to the bacterial cell wall.

4. Bordetella pertussis Specimen Collection
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Bordetella pertussis Specimen Collection
Nasopharyngeal swab or aspirate
Plate at bedside
Regan-Lowe media
Bordet-Gengou media
Methicillin or cephalexin added to media to inhibit normal flora
Make smear for DFA screening
The specimen of choice for recovering Bordetella pertussis is a Nasopharyngeal swab or aspirate. Culturing these specimens is best done at bedside. Special media such as Regan-Lowe or Bordet-Gengou must be used. Regan-Lowe media contains charcoal agar with 10% horse blood and cephalexin. Bordet-Gengou media contains potato agar with glycerol and sheep blood. Methicillin or cephalexin can be added to inhibit normal flora.
In addition two smears are made at bedside – one for the gram stain and the other for DFA screening. The number of organisms present in the secretions limits the sensitivity of the DFA screen so a culture must always be done.
If the specimen must be transported, it is recommended that the swabs be placed in liquid Regan-Lowe media.

5. Bordetella pertussis Growth Characteristics
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Bordetella pertussis Growth Characteristics
35ºC, 5-10% CO2 for at least 7 days
Colony morphology
No growth on BAP & MAC
Bordet-Gengou
Regan-Lowe
Bordetella pertussis is a fastidious obligate aerobe, so upon arrival in the lab, the media should be incubated in a humid environment at 35ºC with 5-10% CO2 for at least 7 days.
Growth generally occurs in 3-5 days. Bordetella pertussis will not grow on BAP and MAC.
On Bordet-Gengou, colonies will be slightly beta hemolytic with smooth, shiny colonies resembling mercury droplets.
On Regan-Lowe, colonies are domed and shiny with a white mother-of-pearl opalescence. The picture provided here is of charcoal-based Regan-Lowe media.

6. Bordetella pertussis Identification
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Bordetella pertussis Identification
Gram stain
Small, faintly staining gram-negative coccobacilli
Oxidase +
Nitrate –
Urea –
Nonmotile
To identify Bordetella pertussis several tests are done.  Its Gram stain morphology shows small faintly staining gram negative coccobacilli. It is oxidase positive, nitrate negative, urease negative and nonmotile.

7. Bordetella pertussis Serological Testing
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Bordetella pertussis Serological Testing
Direct fluorescent antibody test
Agglutination methods
DNA probes for direct detection in:
Specimen
Culture confirmation
Bordetella pertussis is usually identified by direct fluorescent antibody testing. One of the specimen slides prepared at bedside is stained with fluorescent-tagged pertussis antibody. If pertussis is present, fluorescing bacteria will be visible.  Agglutination methods are also available. DNA probes are also available for direct detection in the specimen and for culture confirmation.

8. Bordetella pertussis Treatment & Prevention
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Bordetella pertussis Treatment & Prevention
Erythromycin is drug of choice
Vaccination
When treating Bordetella pertussis, Erythromycin is the drug of choice.  Vaccination of children is the best protection.

9. What media is used to culture Bordetella pertussis?
Regan-Lowe media and Bordet-Gengou media. Bordetella pertussis will not grow on BAP or MAC. Methicillin or cephalexin can be added to the media to inhibit normal flora.

10. Francisella tularensis Clinical Significance
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Francisella tularensis Clinical Significance
Causes Tularemia – an acute febrile, HIGHLY INFECTIOUS disease
Acquire by:
Direct contact with infected animals (rabbits)
Bite from an insect
Inhalation of infectious aerosols
Francisella tularensis causes tularemia, an acute febrile, HIGHLY INFECTIOUS disease. It can be acquired in 3 ways: by direct contact with infected animals such as rabbits, a bite from an insect, or by inhalation of infectious aerosols. This past summer we read in the news about three men from Lincoln getting tularemia while mowing a lawn and hitting a rabbit nest.

11. Francisella tularensis Specimen Collection
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Francisella tularensis Specimen Collection
Inflammatory material from infected site
Wear gloves and use biosafety hood
Do not aerosolize or allow contact with skin or mucous membranes
CDC: Biosafety Level 2 pathogen
The specimen of choice should be inflammatory material from the infected site such as a lesion, lymph node aspirate, sputum, or tissue biopsy. It is critical that you wear gloves and perform all work in a biological safety hood. Do not aerosolize or allow specimen to come in contact with your skin or mucous membranes as this organism can penetrate unbroken skin. Be careful!!!

12. Francisella tularensis Specimen Processing
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Francisella tularensis Specimen Processing
Requires cysteine / cystine for growth
Glucose-cystine blood (Francis’) agar
Grows on
Chocolate
BCYE
Modified TM
Francisella tularensis requires cysteine / cystine for growth so the media of choice is Glucose-cystine blood agar (Francis’ agar). Francisella tularensis will also grow on Chocolate agar, buffered charcoal yeast extract (BCYE) and modified Thayer-Martin (MTM) agar.

13. Francisella tularensis Growth Characteristics
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Francisella tularensis Growth Characteristics
Strict aerobic
35°C with 5-10% CO2 for 7 days
Colony morphology
BAP & MAC = No growth
CHOC = Small, gray alpha-hemolytic colony at 2-5 days
All culture plates are incubated at 35°C with 5-10% CO2 for 7 days Francisella is a strict aerobe. Colony morphology shows no growth on BAP and MAC. The Choc agar has small, gray alpha hemolytic colonies at 2-5 days.

14. Francisella tularensis Identification
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Francisella tularensis Identification
Pale staining gram negative coccobacilli
Oxidase –
Catalase – to weak +
Glucose fermenter
Nonmotile
Francisella tularensis’ gram stain morphology is a pale staining gram negative coccobacillus. Biochemically it is oxidase negative, Catalase negative or weakly positive, a glucose fermenter, and Non-motile. Please NOTE that Francisella tularensis is usually identified by serologically due to the risk of laboratory acquired infection. Biochemical identification is usually performed just in reference laboratories.

15. Francisella tularensis Serological Testing
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Francisella tularensis Serological Testing
Most cases diagnosed serologically
DFA tests may be performed on specimen
ELISA and agglutination tests
Four-fold rise in titer is diagnostic
Single titer of >=1:160 is presumptive
Most cases of Francisella tularensis is diagnosed serologically. Direct Fluorescent Antibody testing may be performed on the specimen. In addition, antibody titers can be determined by ELISA and agglutination tests. A four-fold rise in titer between acute and convalescent specimen is considered diagnostic, and a single acute phase titer of 1:160 or greater is considered presumptive.

16. Francisella tularensis Treatment & Prevention
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Francisella tularensis Treatment & Prevention
Streptomycin is drug of choice
Streptomycin is drug of choice for treatment of tularemia.

17. What substance is required in culture media in order to grow Francisella?
Cysteine / Cystine

18. What is the best method for determining if a patient has Tularemia and Why?
Serological testing is best. To actually culture the organism in the laboratory has a high risk for laboratory personnel becoming infected.

19. Legionella species Clinical Significance
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Legionella species Clinical Significance
Legionnaires’ disease
Pontiac Fever
Transmission: inhalation of the organism in aerosols
Legionella pneumophila serogroup 1
Legionella species causes two different forms of disease: The first is Legionnaires’ disease which is characterized by malaise, myalgia with rapid onset of a dry cough and fever, and eventual development of a pneumonia. This illness is fatal in 15-30% of cases if not treated. The second is disease is Pontiac Fever which is characterized by fever, headache, myalgia and malaise but lacks symptoms of pneumonia and is not fatal.
These two diseases occurs most frequently in men, cigarette smokers, people with underlying disease, immunocompromised patients, and people who drink alcohol. Legionella is a major cause of nosocomial pneumonia.
The organism exists in natural or man-made water systems and in the soil. It is transmitted by inhalation of aerosols containing the organism. Legionella pneumophila serogroup 1 causes most human infections.

20. Legionella species Specimen Collection
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Legionella species Specimen Collection
BAL, BW, lung biopsy, pleural fluid
Avoid aerosolization
Transport ambient temperature
Requires cysteine and iron salts for growth
Buffered Charcoal Yeast Extract agar
Selective media: BCYE + antibiotics
Appropriate specimens when suspecting Legionella include Bronchial Aveolar Lavage, bronchial washing, lung biopsy, and pleural fluid. It is important to avoid aerosolization of the specimen, and to transport specimen at ambient air and room temperature.
Buffered Charcoal Yeast Extract agar (BCYE) is most widely used media for isolating Legionella. The organism requires cysteine and iron salts for growth. When culturing contaminated specimens, you can use BCYE agar with antibiotics added which is selective for Legionella and inhibits normal respiratory flora.

21. Legionella species Growth Characteristics
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Legionella species Growth Characteristics
Aerobic
35°C in 5-10% CO2 for 10 days
Colony morphology
BAP & MAC = no growth
CHOC = grows slowly
BCYE = convex, grayish white, glistening with an entire edge at 2-4 days
The media is incubated at 35°C in 5-10% CO2 with increased humidity for 10 days. Legionella is an aerobic organism and will usually take 2-4 days to grow. The organism will not grow on BAP & MAC, and may grow very slowly on CHOC.
Colony Morphology on BCYE is a convex, grayish white, glistening colony with an entire edge at 2-4 days. When viewed with dissecting microscope they have a "ground glass" appearance with a pink or blue-green tint.

22. Legionella species Identification
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Legionella species Identification
Thin, faintly staining short to filamentous GNR
Oxidase wk +
Catalase wk +
Non-”F”
Non-”O”
Motile:polar flagella
Legionella’s Gram stain morphology is a thin, faintly staining short to filamentous gram negative rod. It is oxidase and catalase weakly positive, Gelatin positive for most species, motile by polar flagella and they do not oxidize or ferment carbohydrates. Some species fluoresce under UV light and color varies depending on the species.

23. Legionella species Serological Testing
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Legionella species Serological Testing
Specimen screen & Isolate ID
DFA stain and DNA probe
IFA test of choice (serum)
Four-fold rise in titer to at least 1:128
A direct screen of the specimen can be done with either a Direct Fluorescent Antibody stain or DNA probe. In addition, the isolate on BCYE can be identified with DFA or DNA probe.
The Indirect Fluorescent Antibody is the test of choice. A four-fold rise in titer to at least 1:128 must be demonstrated in serum. The acute specimen must be drawn within 7 days of onset and the convalescent specimen collected 3-5 weeks after onset.

24. Legionella species Treatment and Prevention
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Legionella species Treatment and Prevention
Susceptibility testing not routinely performed
Drug of choice: Erythromycin alone or with Rifampin
Susceptibility testing is not routinely performed. The drug of choice is Erythromycin by itself or along with Rifampin.
Prevention involves proper filtering and cleaning of air handling systems, whirlpools, air conditioners, and water treatment systems.

25. What substance is required in culture media in order to grow Legionella?
Cysteine and iron salts.

26. What populations are most prone to Legionella infections?
Men, cigarette smokers, people with underlying disease, immunocompromised/immunosuppressed patients, people who drink alcohol and nosocomial infections.

27. Fastidious GNR Summary
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Fastidious GNR Summary
Looked at several organisms
Clinical significance
Specimen collection, transport & processing
Growth characteristics & identification
Serological testing
Treatment and prevention
So, in summary, we have looked at several fastidious gram-negative rods including Bordetella pertussis, Brucella species, Capnocytophaga species, Eikenella corrodens, Francisella tularensis, Legionella species, and the HACEK group. They all are know to cause serious diseases and most have special specimen collection, transport and processing requirements. They also have special growth requirements and characteristics. Besides traditional biochemical testing to identify these organisms, serological testing is often the method of choice to identify organisms in a specimen, serum, or isolate on a culture plate. Susceptibility testing is generally not done because of the special growth requirements, so treatment is based on drugs that have a good track record with previous patients.

28. Who am I? Bordetella pertussis Reagin-Lowe media Gram Stain
Causes Whooping Cough
Bordetella pertussis

29. Who am I?
BCYE agar
Gram Stain
Causes Pontiac Fever
Legionella species