Fastidious Gram Negative Rods Blood Culture Unit Please click audio icon to hear Carol’s narration Fastidious Gram Negative Rods Blood 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)

General Information Fastidious Faint staining Gram Negative Rods Click icon for audio 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.

Brucella species Clinical Significance Click icon for audio Brucella species Clinical Significance Causes Brucellosis / Undulant fever / Malta fever Transmission via: Direct contact with infected animals Ingestion of contaminated meat or dairy products Inhalation of the aerosolized organism Facultative intracellular organism There are four species pathogenic to man: Brucella abortus (cattle), Brucella canis (dogs), Brucella melitensis (goats), and Brucella suis (pigs). Brucella species causes brucellosis, also known as undulant fever or Malta fever, which is a relapsing febrile illness. It is transmitted three ways: via direct contact with infected animals, by ingestion of contaminated meat or dairy products, or by inhalation of the aerosolized organism. Incidence in the US has declined due to vaccination of animals and pasteurization processes. Imported cheeses and candies have been implicated in US cases. Brucella is a facultative intracellular organism and is able to exist in both intracellular and extracellular environments. It can even survive within phagocytic cells.

Brucella species Specimen Collection Click icon for audio Brucella species Specimen Collection Blood, bone marrow or tissue Risk of lab acquired infection Specify on requisition when suspecting Brucella BAP, CHOC, BCYE, Blood Culture broth Specimens of choice are blood, bone marrow or tissue. There is a risk of laboratory acquired infection so it is very important to always work in a biological safety hood when handling specimens and cultures. The test requisition must specify when the physician is suspecting Brucella. Brucella grows on CHOC, BAP, Buffered Charcoal Yeast Extract agar (used for isolation of Legionella), and blood culture media.

Brucella species Growth Characteristics Click icon for audio Brucella species Growth Characteristics 35C, 5-10% CO2 for 21 days Colony morphology Growth at 7 days BAP = smooth glistening, translucent colonies that become brown with age Culture media should be incubated at 35ºC in 5-10% CO2 with a humid atmosphere. The organism is aerobic so any high levels of CO2 can prevent growth. The organism is very slow to grow so hold all cultures for at least 21 days. Growth usually occurs at 7 days. Colony morphology on BAP shows smooth glistening, translucent colonies that become brown with age.

Brucella species Identification Click icon for audio Brucella species Identification Gram stain: tiny, faint staining gram negative coccobacilli Oxidase +, Catalase + Nitrate + Glucose oxidizer Nonmotile Additional tests to speciate Gram Morphology for Brucella shows tiny faint staining gram negative coccobacilli. They are oxidase positive, Catalase positive, Nitrate positive, a Glucose oxidizer and Non-motile. Additional tests must be performed to speciate the Brucella. Your lecture notes provide this information as well as Table 41-2 on page 489 of your textbook.

Brucella species Serological Testing Click icon for audio Brucella species Serological Testing Tube agglutination test Single titer of >=1:160 Fourfold rise in titer between acute and convalescent specimens Does not detect Brucella canis Serological testing is often used with a tube agglutination test most commonly done. A single titer of >=1:160 or a fourfold rise in titer between acute and convalescent specimens is considered significant. This test will not detect Brucella canis.

Brucella species Treatment and Prevention Click icon for audio Brucella species Treatment and Prevention Doxycycline and rifampin for 6 weeks Recommended treatment is doxycycline and rifampin for 6 weeks.

How long must cultures be held when Brucella is suspected? Culture plates and media must be held for at least 21 days because Brucella is a slow grower.

Eikenella corrodens Clinical Significance Click icon for audio Eikenella corrodens Clinical Significance Normal flora of mouth, respiratory & GI tracts Opportunistic pathogen Associated with: Dental / periodontal and head / neck infections / abscesses Human bite wounds Septicemia following tooth extraction Endocarditis There is only one species with the Genus Eikenella and it is Eikenella corrodens. This organism is normal flora of mouth, respiratory tract and GI tract of man. It is an opportunistic pathogen and associated with several diseases: dental/periodontal and head/neck infections/abscesses, human bite wounds, septicemia following tooth extraction and endocarditis.

Eikenella corrodens Specimen Collection Click icon for audio Eikenella corrodens Specimen Collection Aerobic Requires hemin BAP, CHOC Eikenella corrodens has no special specimen collection requirements. It needs hemin in the media for growth so a BAP and CHOC plates can be used.

Eikenella corrodens Growth Characteristics Click icon for audio Eikenella corrodens Growth Characteristics Tiny colonies at 48 hrs Colony morphology BAP “Pits" the agar Pale yellow pigment Greening around colony Bleach odor MAC – no growth Eikenella corrodens grows on a BAP and CHOC at 48 hours as tiny colonies. The MAC has no growth. Colonies on BAP will "pit" (corrode) the agar and are often sunk into small craters in the agar. The colonies will usually have a pale yellow pigment and may have greening around colony. Upon first opening the culture plate, you may notice a characteristic bleach odor.

Eikenella corrodens Identification Click icon for audio Eikenella corrodens Identification Small, slender Gram Negative Bacilli Oxidase + Catalase – Glucose non-oxidizer (asaccharolytic) Nitrate + Nonmotile To identify Eikenella corrodens several tests are performed.  Its Gram stain morphology shows small, slender GNR. Biochemically it is oxidase positive, Catalase negative, a Glucose non-oxidizer (asaccharolytic), Nitrate positive and non-motile.

Eikenella corrodens Treatment and Prevention Click icon for audio Eikenella corrodens Treatment and Prevention Susceptibility testing not routinely done Treat with: Penicillins 3rd generation cephalosporins Tetracycline Quinolones Susceptibility testing is not routinely performed on Eikenella corrodens. Treatment is with penicillins, 3rd generation cephalosporins, tetracycline, and quinolones.

What are the key identifying characteristics for Eikenella corrodens? It pits the agar and has a bleach smell on BAP at 48 hours, it will not grow on MAC, it is oxidase positive, catalase negative, glucose N“F”/N“O”, and nitrate positive.

Haemophilus aphrophilus Actinobacillus actinomycetemcomitans Click icon for audio HACEK Group Organisms Haemophilus aphrophilus Actinobacillus actinomycetemcomitans Cardiobacterium hominis Eikenella corrodens Kingella kingae The organisms included in the HACEK Group are … As you can see, the HACEK group gets its name from the beginning letters for each of the organisms.

HACEK Group Clinical Significance Click icon for audio HACEK Group Clinical Significance Subacute bacterial endocarditis Blood Cultures Subculture to various enriched media and hold for extended time beyond 1 week BCYE Organisms in the HACEK Group are known to cause infective endocarditis. They are usually isolated from blood cultures. It is important to hold the blood cultures for an extended period beyond 1 week and to make blind subcultures of the blood culture broths to several enriched media, including buffered charcoal-yeast extract media. As you can see, these organisms are fastidious in nature.

What organisms are included in the HACEK group? Haemophilus aphrophilus Actinobacillus actinomycetemcomitans Cardiobacterium hominis Eikenella corrodens Kingella kingae

Fastidious GNR Summary Click icon for audio 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.

Who am I? Brucella species BAP, growth appears at 7 days Gram Stain Causes Undulant Fever Brucella species