University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln U.S. Department of Veterans Affairs Staff Publications U.S. Department of Veterans Affairs 1994 Haemophilus parainfluenzae Endocarditis: Application of a Molecular Approach for Identification of athogenicP Bacterial Species Kamal A. Hamed Stanford University School of Medicine Philip R. Dormitzer Stanford University School of Medicine Catherine K. Su Stanford University School of Medicine David A. Relman Stanford University School of Medicine, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/veterans Hamed, Kamal A.; Dormitzer, Philip R.; Su, Catherine K.; and Relman, David A., "Haemophilus parainfluenzae Endocarditis: Application of a Molecular Approach for Identification of athogenicP Bacterial Species" (1994). U.S. Department of Veterans Affairs Staff Publications. 2. https://digitalcommons.unl.edu/veterans/2 This Article is brought to you for free and open access by the U.S. Department of Veterans Affairs at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in U.S. Department of Veterans Affairs Staff Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 677 Haemophilusparainfluenzae Endocarditis: Application of a MolecularApproach for Identificationof PathogenicBacterial Species Kamal A. Hamed, Philip R. Dormitzer, Catherine K. Su, From the Division of Infectious Diseases and GeographicMedicine, and David A. Relman Department of Medicine, Stanford University School of Medicine, Stanford;and the Departmentof VeteransAffairs Medical Center, Palo Alto, California Haemophilus parainfluenzae is both a human oropharyngeal commensal bacterium and a cause of serious invasive disease. The fastidious growth characteristics of this organism and the poor specificity of traditional methods for species identification are likely to have led to inaccura- cies in the diagnosis of infections caused by H. parainfluenzae and related organisms. We report a case of H. parainfluenzae endocarditis in which confusion related to microbial identification was resolved by the analysis of 16S ribosomal RNA sequences. Rapid identification was facilitated by amplification of 16S ribosomal DNA directly from cultured cells with use of the polymerase chain reaction and by direct DNA sequence determination of the amplified product. This proce- dure is potentially useful for the identification of fastidious bacterial pathogens by reference laboratories. Haemophilusparainfluenzae is a common inhabitant of lation or identification,especially of Haemophilusparaphro- the upperrespiratory tract as well as an occasionalpathogen. philus and H. parainfluenzae.These organismsremain diffi- In one study, it was isolated from the throats of all of 10 cult to cultivatein the laboratoryand usually requireat least healthy childrenand all of 10 healthy adultsand constituted 1 week for identificationto the species level. Theiridentifica- 74% of cultivated pharyngealHaemophilus organisms [1]. tion has been based on metabolic or biochemical features Among Haemophilusspecies, this organism is the second that may be variableor nonspecific [5]. most frequentlyisolated pathogen after Haemophilus influen- Species and, in some instances, strains can be reliably zae. Reported disease syndromescaused by H. parainfluen- identifiedby the analysis of certain specific gene sequences zae include endocarditis,meningitis, pharyngitis, epiglottitis, -in particular,those of the ribosomalRNA (rRNA) genes. otitis media, dental abscess, conjunctivitis, brain abscess, This type of approachhas been appliedclinically to the iden- pneumonia, empyema, septicemia, septic arthritis, osteo- tificationof"unculturable" microbial pathogens and fastidi- myelitis, soft-tissueabscess, peritonitis,hepatic abscess, and ous microorganismssuch as the mycobacteria[6-9]. In this urinarytract infection [2, 3]. paper,we use a case of endocarditisto illustratehow a 16S Endocarditiscaused by Haemophilusspecies accounts for rRNA-based approachcan be used to expeditiouslyresolve more than half of all cases caused by a group of fastidious confusion regardingthe identificationof H. parainfluenzae. coccobacillaryorganisms known as the HACEKgroup. The other in this are Actinobacillus organisms group actinomyce- Materials and Methods temcomitans,Cardiobacterium hominis, Eikenella corrodens, and Kingellakingae. HACEK organisms, in turn,cause more Determinationof 16S rRNAsequence. A partialsequence than half of all cases of gram-negativebacterial endocarditis of the 16S rRNA gene of the bacterialisolate-referred to as [4]. It is likely that many cases of endocarditiscaused by UNK-from the case describedbelow was obtained as fol- these organismshave been classified as culture-negativeor lows. The bacterialcell mass fromseveral minute colonies of attributedto the wrong species because of difficultiesin iso- a blood-bottlesubculture was digestedovernight at 55?C in 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1%Laureth-12 (Mazer Chemicals, Gurnee, IL), and 0.2 mg of proteinase K/mL (BoehringerMannheim, Indianapolis),as previously Received 5 November 1993; revised 18 April 1994. described After inactivationof the K at 94?C Financial support:Lucille P. MarkeyCharitable Trust (to D.A.R., a Lu- [6]. proteinase cille P. MarkeyBiomedical Scholar); Stanford PMGM Director'sResearch for 10 minutes, DNA from 1-/gLand 10-/1laliquots of the Fund, sponsoredby SmithKlineBeecham (to D.A.R.); and StanfordDepart- lysatewas amplifiedby the polymerasechain reaction(PCR) ment of Microbiologyand ImmunologyNational ResearchService Award with use of the bacterial 16S rRNA T32-AI07328 (to P.R.D.). broad-range primers Reprintsor correspondence:Dr. David A. Relman, Palo Alto Veterans 8FPLand 806R [10, 1 1]. The PCR productswere separated AffairsMedical Center, 154T, 3801 MirandaAvenue, Palo Alto, California by agarosegel electrophoresisand purifiedwith the Magic 94304. PCR Preps System (Promega Corporation,Madison, WI). ClinicalInfectious Diseases 1994;19:677-83 The DNA were with ? 1994 by The Universityof Chicago.All rightsreserved. purified fragments directly sequenced 1058-4838/94/1904-0006$02.00 the Taq polymerase-basedfmol sequencing system (Pro- 678 Hamedet al. CID 1994;19(October) mega). The sequencingprimers 50F (5'AACACATGCAAG- time yielded CSF containing6 red blood cells/mm3,9 leuko- TCGAACG3')and 512R (5'CGAATTCCCGCGGCTGCT- cytes/mm3(100% monocytes), 27 mg of protein/dL,and 58 GGCACGGA3') were based on conserved bacterial 16S mg of glucose/dL; the blood glucose level was 85 mg/dL. rRNA sequences [11, 12]. The sequencing reactions were Gram stainingof the CSF revealedno organisms. chased with terminal deoxynucleotidyltransferase(Gibco On admission the patient was lethargic and somewhat BRL, Gaithersburg,MD), and 35S-labeledsequencing prod- confused;her temperaturewas 39?C, her blood pressurewas ucts were analyzed by separationon buffer gradient poly- 100/68 mm Hg, and her pulse was 108/min, with orthostatic acrylamide-ureagels and autoradiography[13]. changes. Her fundi were unremarkableand her neck was Multiple-sequencealignment was performedwith Pileup, supple. Examinationof her mouth revealedgood dental hy- a programin the GeneticsComputer Group Sequence Analy- giene with multiple eruptingwisdom teeth. A grade 1/6 sys- sis SoftwarePackage, version 7.1.1 [14]. Pileup uses a simpli- tolic murmurwas heard by one examiner. The lungs were fication of the progressivealignment method to createalign- clear to auscultationand percussion. Hepatosplenomegaly ments and a dendrogram[15]. The sequences of 16S rRNA was detected. The patient was oriented to time, place, and genes from membersof the family Pasteurellaceae,including person, and she had no focal neurologicalfindings. The rest a representativeof each subclusterdefined by Dewhirstet al. of the resultsof her physicalexamination were normal. [16], and from Escherichiacoli were obtainedfrom GenBank Initiallaboratory findings included a peripheral-bloodleu- (Bethesda,MD) and used as a data set for the alignment.The kocyte count of 17,000/mm3,with 65%polymorphonuclear species of origin for these sequences(as listed by Dewhirstet cells and 22%band forms;a hematocritof 32.0%;a platelet al. [16]), the straindesignations, and the GenBankaccession count of 84,000/mm3; and the following serum levels: so- numbers are as follows: A. actinomycetemcomitans,ATCC dium, 135 mmol/L; potassium, 2.7 mmol/L; chloride, 95 29522, M75036; H. aphrophilus,ATCC 33389T, M75041; mEq/L;bicarbonate, 30 mEq/L;albumin, 2.7 g/dL; and lac- H. influenzae,ATCC 33391, M35019 and M59433; Actino- tate dehydrogenase,952 IU/L. Serum levels of creatinine, bacillus seminis, ATCC 15768, M75047; Pasteurellaspe- alkaline phosphatase,aspartate aminotransferase, and ala- cies, CCUG 18782, M75055; Pasteurellamultocida, NCTC nine aminotransferasewere within normallimits. Urinalysis 10322, M35018 and M59769; Haemophilus parasuis, revealedmicroscopic hematuria. A chest roentgenogramwas NCTC 4557, M75065; Actinobacillussuis, ATCC 15557, normal. Blood was drawn for cultures,and therapywas be- M75071; Actinobacillusureae (listed by GenBankas Pasteur- gun with parenteralbroad-spectrum antibiotics. ella ureae), Henrikson 3520/59T, M75075; Actinobacillus On the second day of hospitalization,the patient devel- species (listed by GenBank as Actinobacilluscapsulatus), oped photophobia,increased lethargy, and disorientationto CCUG 19799, M75067; Actinobacilluspleuropneumoniae,