Article 364 CASES IN CLINICAL MICROBIOLOGY 1 Clock Hour Case Eighteen: Infection or Environment? Anamarija Morovic, Ashley Bowman, Connie Meeks, and Joel Mortensen Background EDITOR’S NOTE: BEFORE reading the Case Cystic fibrosis is an autosomal recessive disease, Follow-up and Discussion below, study the Case resulting from mutations in the cystic fibrosis trans- Description on page 60 of this issue, and formu- late your own answers to the questions posed. membrane conductance regulator. Disease involves multiple systems, and is characterized by production of sticky, dehydrated mucus that lines epithelial cells Case Follow-up and Discussion of the respiratory tract, predisposing the patient to in- The patient returned to the Clinic after completion fections by a variety of opportunistic pathogens. The of antibiotic therapy, reporting significant improve- most commonly isolated pathogen is Pseudomonas ment of symptoms. Values of his pulmonary function aeruginosa (80%). Other more infrequent species in- tests returned to baseline (FEV1-102%). clude Burkholderia cepacia, Achromobacter xy- Because the patient had not previously had B. losoxidans and Stenotrophomonas maltophilia. The cepacia, the specimen was sent for final identifica- incidence of Herbaspirillum colonization/infection tion to the Cystic Fibrosis Foundation Burkholderia in patients with cystic fibrosis is uncertain. Spilker, cepacia Research Laboratory and Repository in Ann et al., report incidence of less than 3%, which is sim- Arbor, MI. Sequencing of 16s rRNA was performed ilar to the incidence of Burkholderia cepacia. This and the organism was identified as a Gram-negative study included 28 cystic fibrosis patients infected bacillus with the highest identity to Herbaspirillum with Herbaspirillum, who ranged in age from 20 species. The isolated was tested for antimicrobial sus- months to 59 years (average 17 years). Most of the ceptibility using the ETest method (bioMerieux, patients had transient respiratory tract colonization France) The isolate was found to be susceptible to the with Herbaspirillum, while two patients had bac- Anamarija Morovic, majority of antimicrobial agents tested (see Table 1). teremia and chronic respiratory tract infection1. MD, is a pathology Herbaspirillum species are classified as members resident in the Depart- ment of Pathology, of the class Betaproteobacteria; order, Burkholderi- University of Cincin- ales; family, Oxalobacteracea. Members of this order TABLE 1: MIC values for Herbaspirillum isolate nati; include a number of genera containing opportunistic Connie Meeks, RN, is Antimicrobial agent MIC (μg/ml) pathogens of CF patients, e.g., Achromobacter, Aci- a nurse practitioner in dovorax, Alcaligenens, Burkholderia, Comomonas, the Department of Amikacin 12 2 Pulmonary Medicine; Cupriavidus, Pandoarea and Ralstonia. The genus Ashley Bowman, Aztreonam 16 currently consists of 10 species: Herbaspirillum au- MT(ASCP), is a med- Colistin >256 totrophicum, Herbaspirillum chlorophenolicum, ical technologist in the Herbaspirillum frisingense, Herbaspirillum hiltneri, Diagnostic Infectious Doxycycline 0.5 Herbaspirillum huttiense, Herbaspirillum lusitanum, Diseases Testing Lab- oratory of the Depart- Cefepime 0.19 Herbaspirillum putei, Herbaspirillum rhisosphaer- ment of Pathology and ae, Herbaspirillum rubrisubalbicans, and Herbaspir- Laboratory Medicine; Tigecycline 0.75 illum seropedicae1. and Ciprofl oxacin 0.75 Human infections with Herbaspirillum species are Joel E. Mortensen, rare; however, when present, the organisms most Ph.D., is the Director Meropenem 0.125 of the Diagnostic In- commonly cause lung infections in patients with cys- fectious Diseases Test- Piperacillin-tazobactam <0.016 tic fibrosis. Herbaspirillum was first isolated from the ing Laboratory of the respiratory tract of a patient with cystic fibrosis in Department of Pathol- Ticarcilin-clavulanate 2 2000. Since then, there have been less than 30 report- ogy and Laboratory ed cases, almost all isolated by the Burkholderia Medicine, Cincinnati Tobramycin 4 Children's Hospital, cepacia Research Laboratory and Repository (Uni- Cincinnati, Ohio Ceftazidime 1 versity of Michigan, Ann Arbor).1,3 68 April 2010 • Continuing Education Topics & Issues Reservoir Bacteria of the genus Herbaspirillum are mostly associated with plants, either as an asymptomatic bacteria or as causal agents of mild disease. They have been isolated from plants found in the species of the family Gramineae such as rice, wild rice, Sorghum bicolor, Miscanthus sinensis and Pennise- tum purpureum. They are also associated with di- cotyledoneous plants and can be found in the root nodules of the legume Phaseolus vulgaris. Addition- ally, they have been isolated from roots and stems of bananas and pineapples, as well as from sugarcane.1,4,5 The natural reservoir for a number of opportunistic pathogens, such as Herbaspirillum, Burkholderia, Ochronobactrum, Pseudomonas, Ralstonia, and Stenotrophomonas, is the plant rhizosphere. The rhi- Figure 2. Growth of the organism on Chocolate o zosphere is the layer of soil influenced by root me- agar following incubation at 35 C in 5% CO2 for tabolism. It is a nutrient-rich niche for microorgan- 24 hours. isms which fulfill important ecological functions and are responsible for plant growth and health. Many of Laboratory identification these bacteria are able to grow at 37°C, allowing them Herbaspirillum species are gram-negative, to survive in humans. Human exposure to plants can motile, nitrogen fixing bacteria (Figure 1). They lead to colonization and disease in susceptible indi- are reported to grow well on B. cepacia selective 1 viduals, such as patients with cystic fibrosis, AIDS, agar (BCSA). This isolate also grew well on older patients with chronic diseases, patients on long- chocolate agar. On chocolate agar, the colonies term antimicrobial therapy and immuno-suppressed are small, and whitish-grey (Figure 2). patients. Because of their ecological niche and the ex- Herbaspirillum can be isolated aerobically in posure to antibiotic substances in the rhizosphere, iso- Mueller-Hinton broth that is supplemented with lates of Herbaspirillum are often very resistant to 1.6% (wt/vol) agar and incubated at 32°C for conventional antimicrobial agents used in human 24-48 h. Organisms are oxidase and ONPG pos- medicine.6 itive, and lysine decarboxylase negative. They variably ferment lactose. Definitive identifica- Clinical presentation tion is best achieved by 16S rRNA-targeted PCR Members of Herbaspirillum species are rare amplification, sequencing and editing, which human pathogens. They most commonly cause lung are then compared to sequences available in the infections in patients with cystic fibrosis, but organ- NCBI GenBank bacterial DNA database. Spilker, isms have been sporadically recovered from various et.al., examined and reported initial identifica- human sources including wounds, gastric specimens, tion reported by various test systems, using 16S feces, urine, eye and ear samples, and blood.1 There rRNA sequencing as the gold standard. These has been one reported case of lower extremity celluli- test systems included MicroScan, RapidNF, tis and bacteriemia caused by Herbaspirillum sero- Vitek, API NF, Phoenix and Biochem. Nineteen pedicae in a patient with cirrhosis.3 out of twenty-eight referred isolates were ini- tially identified as Burkholderia cepacia com- plex, four as Ralstonia species and five were not identified to the species level, thus reinforcing the need for molecular methods when attempt- ing to definitively identify this genus.1 Antimicrobial susceptibilities There is little published data concerning antimi- crobial susceptibility patterns for Herbaspirillum species in human isolates, which is likely due to the rarity of this organism. Susceptibilities of our isolate are listed in Table 1. Conclusions Herbaspirillum species are opportunistic plant Figure 1. Gram stain of the organism. pathogens which may be increasingly identified from Continuing Education Topics & Issues • April 2010 69 the respiratory cultures of patients with cystic fibro- References sis. The majority of the reported patients had only 1. Spilker T, Uluer AZ, Marty FM, Yeh WW, Levison JH, Van- transient respiratory colonization, but chronic respi- damme P, LiPuma JJ. Recovery of Herbaspirillum species from persons with cystic fibrosis. J Clin Microbiol. 2008; ratory infection and bacteriemia have been observed. 46:2774-2777. Our patient experienced a significant decrease in res- 2. J.P. Euzéby: List of Prokaryotic names with Standing in piratory function due to infection with Herbaspiril- Nomenclature. http://www.bacterio.cict.fr lum, and successfully recovered after administration 3. Tan MJ, Oehler RL. Lower extremity cellulitis and bacteriemia of antimicrobial agents. with Herbaspirillum seropedicae associated with aquatic ex- posure in a patient with cirrhosis. Infect Dis Clin Pract. 2005; This organism is commonly misidentified by com- 13:277-279. mercial test systems as Burkholderia cepacia com- 4. Schmid M, Baldani JI, Hartmann A. The genus Herbaspiril- plex. This mistake can have an enormous impact on lum. Prokaryotes. 2006; 5:141-150. patients with cystic fibrosis. Infection with Burk- 5. Baldani JI, Baldani VLD, Seldin L, Dobereiner J. Characteri- holderia cepacia complex is considered an absolute zation of Herbaspirillum seropedicae gen. nov., sp. nov., a root-associated nitrogen-fixing bacterium. Int J Syst Bacteriol. contraindication to lung transplantation,