1 Fastidious Gram Negative Rods Blood Culture Unit Please click audio iconto hear Carol’s narrationFastidious Gram Negative Rods Blood Culture UnitHi 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 TechnologyCarol Larson MSEd, MT(ASCP)
2 General Information Fastidious Faint staining Gram Negative Rods Click icon for audioGeneral InformationFastidiousComplex / extensive nutrient requirementsFaint staining Gram Negative RodsSafranin counterstain for >2 minutesSubstitute carbolfuschin for safraninSerological testing usefulFastidious 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 Brucella species Clinical Significance Click icon for audioBrucella species Clinical SignificanceCauses Brucellosis / Undulant fever / Malta feverTransmission via:Direct contact with infected animalsIngestion of contaminated meat or dairy productsInhalation of the aerosolized organismFacultative intracellular organismThere 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.
4 Brucella species Specimen Collection Click icon for audioBrucella species Specimen CollectionBlood, bone marrow or tissueRisk of lab acquired infectionSpecify on requisition when suspecting BrucellaBAP, CHOC, BCYE, Blood Culture brothSpecimens 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.
5 Brucella species Growth Characteristics Click icon for audioBrucella species Growth Characteristics35C, 5-10% CO2 for 21 daysColony morphologyGrowth at 7 daysBAP = smooth glistening, translucent colonies that become brown with ageCulture 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.
6 Brucella species Identification Click icon for audioBrucella species IdentificationGram stain: tiny, faint staining gram negative coccobacilliOxidase +, Catalase +Nitrate +Glucose oxidizerNonmotileAdditional tests to speciateGram 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.
7 Brucella species Serological Testing Click icon for audioBrucella species Serological TestingTube agglutination testSingle titer of >=1:160Fourfold rise in titer between acute and convalescent specimensDoes not detect Brucella canisSerological 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.
8 Brucella species Treatment and Prevention Click icon for audioBrucella species Treatment and PreventionDoxycycline and rifampin for 6 weeksRecommended treatment is doxycycline and rifampin for 6 weeks.
9 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.
10 Eikenella corrodens Clinical Significance Click icon for audioEikenella corrodens Clinical SignificanceNormal flora of mouth, respiratory & GI tractsOpportunistic pathogenAssociated with:Dental / periodontal and head / neck infections / abscessesHuman bite woundsSepticemia following tooth extractionEndocarditisThere 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.
11 Eikenella corrodens Specimen Collection Click icon for audioEikenella corrodens Specimen CollectionAerobicRequires heminBAP, CHOCEikenella corrodens has no special specimen collection requirements. It needs hemin in the media for growth so a BAP and CHOC plates can be used.
12 Eikenella corrodens Growth Characteristics Click icon for audioEikenella corrodens Growth CharacteristicsTiny colonies at 48 hrsColony morphologyBAP“Pits" the agarPale yellow pigmentGreening around colonyBleach odorMAC – no growthEikenella 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.
13 Eikenella corrodens Identification Click icon for audioEikenella corrodens IdentificationSmall, slender Gram Negative BacilliOxidase +Catalase –Glucose non-oxidizer (asaccharolytic)Nitrate +NonmotileTo 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.
14 Eikenella corrodens Treatment and Prevention Click icon for audioEikenella corrodens Treatment and PreventionSusceptibility testing not routinely doneTreat with:Penicillins3rd generation cephalosporinsTetracyclineQuinolonesSusceptibility testing is not routinely performed on Eikenella corrodens. Treatment is with penicillins, 3rd generation cephalosporins, tetracycline, and quinolones.
15 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.
16 Haemophilus aphrophilus Actinobacillus actinomycetemcomitans Click icon for audioHACEK Group OrganismsHaemophilus aphrophilusActinobacillus actinomycetemcomitansCardiobacterium hominisEikenella corrodensKingella kingaeThe 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.
17 HACEK Group Clinical Significance Click icon for audioHACEK Group Clinical SignificanceSubacute bacterial endocarditisBlood CulturesSubculture to various enriched media and hold for extended time beyond 1 weekBCYEOrganisms 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.
18 What organisms are included in the HACEK group? Haemophilus aphrophilusActinobacillus actinomycetemcomitansCardiobacterium hominisEikenella corrodensKingella kingae
19 Fastidious GNR Summary Click icon for audioFastidious GNR SummaryLooked at several organismsClinical significanceSpecimen collection, transport & processingGrowth characteristics & identificationSerological testingTreatment and preventionSo, 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.
20 Who am I? Brucella species BAP, growth appears at 7 days Gram Stain Causes Undulant FeverBrucella species