Aerococcus-Like Organism, a Newly Recognized Potential Urinary Tract Pathogen

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1 JOUNAL OF CLINICAL MICOBIOLOGY, My 1991, p /91/5149-5$2./ Copyright X 1991, Americn ociety for Microbiology Vol. 29, No. 5 Aerococcus-Like Orgnism, Newly ecognized Potentil Urinry Trct Pthogen JEN J. CHITENEN,1,2,3* HENIK VIBIT,1 JAN UING,4 AND BENT KONE' Deprtment of Clinicl Microbiology, Bispebjerg Hospitl,' nd Deprtment of Dignostic Bcteriology nd Antibiotics, ttens eruminstitut,3 Copenhgen, nd Deprtment of Clinicl Microbiology, Centrl Hospitl, Hillerod,2 Denmrk, nd Deprtment of Medicl Microbiology, University of Lund, Mlmo Generl Hospitl, Mlmo, weden4 eceived 11 October 199/Accepted 11 Februry 1991 During the lst 1 to 15 yers, Aerococcus-like orgnisms hve been isolted from urinry trct specimens from hospitlized ptients t Bispebjerg Hospitl, Copenhgen, Denmrk, with signs of urinry trct infection, nd single strin hs been deposited t the Ntionl Collection of Type Cultures (NCTC 12142). An dditionl 63 isoltes from the urine specimens of s mny ptients with suspected urinry trct infection hve been identified from seven deprtments of clinicl microbiology in Denmrk. Clinicl informtion indictes tht this orgnism my be involved in urinry trct infection. These strins nd ten previously collected strins, two of which were from blood cultures, were chrcterized phenotypiclly. By compring the results with dt on other grm-positive, ctlse-negtive cocci or coccobcillry orgnisms, the identified orgnisms seem to represent seprte txon. Infections cused by Aerococcus viridns in humns re seldom reported; cses of urinry trct infections (3, 1), bcteremi (6), endocrditis (3, 5, 1, 11, 15), nd meningitis (9) hve, however, been published. During the lst 1 to 15 yers, we hve observed t Bispebjerg Hospitl, Copenhgen, Denmrk, Aerococcus-like orgnisms (ALO) in urinry trct specimens from hospitlized ptients with signs of urinry trct infection. The ALO strins hve been chrcterized phenotypiclly in previous report (2), nd single urine isolte hs been deposited t the Ntionl Collection of Type Cultures, Colindle, United Kingdom (NCTC 12142). ALO produce greening on 5% horse blood gr like the viridns group streptococci, form in clusters like stphylococci s confirmed by microscopy, nd re ctlse negtive. However, the isoltes cnnot be esily ssigned to ny genus or species. We decided to investigte whether ALO lso could be isolted from urinry trct specimens in other lbortories in Denmrk, to further chrcterize ALO isoltes, nd to comment on the txonomic reltionship of ALO isoltes. MATEIAL AND METHOD Collection of strins nd clinicl fetures of ptients. even deprtments of clinicl microbiology in Denmrk were sked to collect five to ten consecutive strins from urinry trct specimens with the following chrcteristics- grm-positive cocci, predominntly in clusters; colonies resembling lphhemolytic streptococci on 5% horse blood gr; ctlse negtive; susceptible to penicillin; nd resistnt to both sulfonmides nd minoglycosides. Ptients. Predisposing conditions, together with symptoms indictive of urinry trct infection (rectl temperture- * Corresponding uthor C, dysuric complints, nd pyuri), were obtined by reviewing the records of the ptients. trins. The recently collected strins of ALO (n = 63) nd 1 other strins, of which eight nd two strins originted from urine specimens nd blood cultures, respectively, were exmined. tndrd culturl conditions. trins were mintined on 5% horse blood gr nd incubted t 35 C in norml tmosphere. The stndrd inoculum used for liquid cultures ws 1 to 2 drops of 24-h Todd-Hewitt broth culture. Growth. Growth ws tested on.4% tellurite gr, on 4% bile gr, on bilyered sheep blood gr (8), in 6.5% NCl broth, nd t different tempertures in trypsin broth (12). Pltes nd tubes were observed for 2 dys. Biochemicl tests. Biochemicl tests were performed s described previously (2), except tht they were supplemented with test for leucine-minopeptidse ctivity (osco Dignostic Tblet [1]). usceptibility testing. Antibiotic susceptibility ws tested by the direct gr diffusion method by using osco Neo- ensitbs on Dnish blood gr. trins re reported s susceptible when zone dimeters were.28 mm for penicillin (5 jjtg), sulfonmides nd netilmicin; >-1 nd 218 mm for 5 nd 7,ug of vncomycin, respectively; nd -15 nd.22 mm for.4 nd 4 IU of bcitrcin, respectively. Zone dimeters less thn the bove-mentioned figures re reported to indicte resistnce. The inhibition zone limits correlte pproximtely to MIC brekpoints for penicillin t.25,ug/ml, for sulfonmides t 64,ug/ml, for netilmicin t 2 plg/ml, nd for vncomycin (7,g) t 4,ug/ml. The bcitrcin tblets nd the 5,ug of vncomycin used re only for dignostic purposes. Determintion of GC moles percent of DNA. The methods for isoltion of DNA nd estimtion of GC moles percent by the therml denturtion method hve been described erlier (13). The vlues given re the mens of four determintions. Downloded from on October 6, 218 by guest

2 15 CHITENEN ET AL. TABLE 1. Prmeters indictive of urinry trct infection in 63 ptients whose urine yielded growth of ALO Prmeter(s) ALO singly No. of ptients ALO in dmixture Without With Without With ctheter' ctheter ctheter ctheter Dysuri fever pyuri Dysuri fever Dysuri pyuri Fever pyuri 1 1 Dysuri 1 1 Fever 1 Pyuri Incontinence 2b No symptoms 1 1 No informtion 4 3 Indwelling urinry ctheter. b In two ptients with congenitl myelomeningocele nd recurrent nonsymptomtic urinry trct infection, the test for pyuri ws not performed. EULT During the study period, 64 ptients suspected of urinry trct infection due to ALO were identified (Tble 1). The orgnism isolted from one of the ptients ws phenotypiclly different from ALO isoltes. A totl of i15 ALO per ml were cultured from the urine specimens of ll the remining 63 ptients, except for four ptients hving 13 to 14 ALO per ml of urine. ALO were grown in pure culture from the urine of 36 ptients, while ALO were cocultured with other bcteri from the urine of 27 ptients (Tble 2). From five ptients with pure culture of ALO repet specimen before tretment lso showed i15 ALO per ml. Ptients. Of the 63 ptients, 34 were femle nd 29 were mle. The medin ge ws 74 yers (rnge, 3 to 97 yers). Forty-one ptients suffered from conditions predisposing them to urinry trct infection (Tble 3). Elevted serum cretinine ws observed in 12 ptients. Twelve ptients hd received sulfonmides within the lst week before specimen smpling, nd one ptient ech hd received trimethoprim, nitrofurntoin, or penicillin. Description of strins. All 73 isoltes were grm-positive cocci, predominntly in clusters nd with tendency to form pirs nd groups of four. All strins grew well on 5% horse blood gr, producing green discolortion of the gr resem- TABLE 2. Bcteri cocultured with ALO from urine of 27 ptients No. of ptients with: Orgnism(s) _15 _14 bcteri/ml bcteri/ml E. coli 1 2 Klebsiell pneumonie 2 Morgnell morgnii 1 Citrobcter freundii 1 Enterobcter species 1 Enterococcus feclis 1 1 Hemolytic streptococci (group C) 1 tphylococcus ureus 1 Corynebcterium species 1.3 orgnisms 5 The two orgnisms isolted were E. coli-like, but exct species identifiction ws not performed. TABLE 3. Conditions predisposing to urinry trct infection found mong 35 ptients whose urine yielded growth of ALO' Predisposing condition No. of ystemic Dibetes mellitus Prkinson's disese... 3 Cncer (bldder, pncres, lung, uterine cervix) Multiple sclerosis... 2 Polyneuropthi... 2 Locl Hyperplsi of the prostte... 4 Indwelling urinry ctheter... 7 Presis of the bldder... 6 Bldder ppillom... 2 Bldder stones Ureterl reflux ix ptients suffered from two conditions predisposing them to urinry trct infection t the sme time. bling tht of viridns group streptococci. The colony dimeter incresed from.5 mm fter 24 h to 1 mm fter 48 h, nd the colonies of ll strins were convex, with well-defined border, nd were shiny nd trnsprent. No growth ws seen on tellurite gr, wheres on bile gr ll strins grew scntily (fter 24 h) to modertely (fter 48 h). All strins grew in 6.5% NCl broth fter 2 dys of incubtion t 37 C. No growth occurred t 1 C, 45 C, or fter the heting of inoculted tubes to 6 C for 3 min; however, when 1-fold greter inoculum ws used, strins grew t 45 C, mking this test of limited vlue for the identifiction of ALO. CAMP rection ws negtive for ll strins. Growth chrcteristics, results of biochemicl tests, nd ntibiotic susceptibilities of strins re shown in Tble 4. For sulfonmides, no zone ws seen t ll, wheres for netilmicin zone dimeter of 2 to 22 mm ws observed for most strins. The gunine-plus-cytosine content of DNA ws estimted for four strins nd ws mol% (men ± stndrd devition). DICUION J. CLIN. MICOBIOL. This investigtion confirms tht strins phenotypiclly identicl to the ALO isoltes previously described (2) cn be found in urinry trct specimens exmined in most lbortories in Denmrk. Few phenotypic chrcteristics (see Mterils nd Methods) re needed to identify these strins. Their detection is, however, dependent on whether microscopy is performed, s they otherwise my esily be mistken for viridns group streptococci. As lso seen previously, dt from ptient records indicte tht ALO possess certin pthogenic potentil in urinry trct infection; most ptients hd clssicl signs of urinry trct infection, recurrences occurred, nd the distribution of ALO in urine specimens ws equl to wht is seen for estblished pthogens such s Escherichi coli nd Klebsiell spp. ALO lso hs the bility to cuse invsive infections, i.e., urosepticemi nd ftl endocrditis (unpublished dt); in both cses, ntibodies ginst ALO isoltes were demonstrted in crossed immunoelectrophoretic ssy. Identifiction of ctlse-negtive, grm-positive coccl nd coccobcillry orgnisms hs received renewed interest Downloded from on October 6, 218 by guest

3 VOL. 29, 1991 UINAY TACT INFECTION AND ALO 151 TABLE 4. Growth chrcteristics, fermenttion rections, direct enzyme test, nd ntibiotic susceptibility for 73 strins of ALO' Chrcteristic ectionb Dy of positive No. of deviting rection strins Hemolysis Production of ctlse Growth on:.4% tellurite 4% bile Growth in 6.5% NCl Growth t: looc 45C urviving 6C fter 3 min CAMP rection Acid production from: Arbinose Glucose (cid/gs) Mltose Lctose ucrose Trehlose Melibiose ffinose licin Esculin Melizitose Inulin Glycogen Mnnitol orbitol Glycerol Production of: P-Glctosidse P-Glucuronidse Pyrrolidonyl minopeptidse Leucine minopeptidse Hydrolysis of: Hippurte Arginine Acetoin production ection with group D ntiserum usceptibility to: Penicillin G ulfonmides Netilmicin Vncomycin (5 jig) Vncomycin (7,ug) Bcitrcin (.4 IU) Bcitrcin (4 IU) From eight deprtments of clinicl microbiology in Denmrk. b, usceptible;, resistnt. I cnty to moderte growth. l- (24 h), (4 h) during the lst severl yers, prtly stimulted by the emergence of vncomycin-resistnt strins. Gener tht hve to be tken into considertion, s pointed out by Fcklm et l. (4), re the following: Aerococcus, Enterococcus, Lctococcus, treptococcus, Gemell, Leuconostoc, nd Pediococcus. In their report, they dvise the use of the tests shown in Tble 5 for differentiting between gener. We hve dded the phenotypic chrcteristics for ALO strins nd lso GC moles percent of gener (14). Our strins showed little phenotypic intervrition nd seem distinct from the other gener of ctlse-negtive, grm-positive cocci. They differ from streptococci by their microscopic ppernce in tht they do not form chins. Further, the following rections re in disgreement between streptococci nd ALO: for Enterococcus spp., streptococcl group D ntigen, bile-esculin, pyrrolidonyl minopeptidse ctivity (PYse), growth t 1 nd 45 C; for treptococcus spp., growth in 6.5% NCl; for Lctococcus spp., growth t 1 C. However, severl rections for treptococcus spp. nd Lctococcus spp., shown in Tble 5, re species intervrible, mking these rections less useful in delineting different gener. In previous study (2) lso using tests other thn those included in this study, we hve compred ALO with treptococcus snguis, treptococcus bovis, treptococcus group Q nd treptococcus group, these strins being esily distinguishble from ech other. By using the scheme estblished by Kilin et l. (7) in their extensive txonomic revision of viridns group streptococci, ALO re lso reltively esily seprted from. snguis, treptococcus nginosus, nd treptococcus mutns; rections of 1 (No growth) 4 ( dy 1-4) 1 (Negtive) 3 (Negtive) Downloded from on October 6, 218 by guest

4 152 CHITENEN ET AL. J. CLIN. MICOBIOL. TABLE 5. Test Tests useful in differentiting ctlse-negtive, grm-positive cocci nd coccobcillry gener of bctefi nd GC moles percent of gener ection of ALO nd strins of the genusb: Enterococcus treptococcus Lctococcus Aerococcus Gemell Pediococcus Leuconostoc Lctobcillus ALO Morphology Cocci - Coccobcilli ods Arrngement Chins Pirs Clusters Gs from glucose V Vncomycin treptococcl ntigen D A-V N - - D group Bile-esculin V V V - V Aminopeptidse ctivity Pyrrolidonyl V V Leucine - - V Growth In 6.5% NCl - V - V V V At 45C V V - At 1C V - Mol% (GC)C 33.5_39d e From humn clinicl isoltes; dpted from Fcklm et l. (4). b Abbrevitions: V, vrible;, susceptible to vncomycin (presence of zone of inhibition);, resistnt to vncomycin (bsence of zone of inhibition). c From Bergey's Mnul of ystemtic Bcteriology. d Enterococcusfecium subsp. cssiflvum: GC = 42 mol%. e A. viridns: GC = mol%. specil interest re esculin hydrolysis; hippurte hydrolysis; Voges-Proskuer's rection; cid production from mltose, lctose, trehlose, inulin, nd slicin; nd,b-glctosidse nd,-glucuronidse production. The type strin of A. viridns produces 3-glctosidse nd PYse but not P-glucuronidse or leucine minopeptidse, rections just opposite to wht is seen for ALO strins. Leucine minopeptidse is lso typiclly produced by Gemell spp. nd pediococci, with which ALO strins hve mjor chrcteristics in common, lthough the following chrcteristics seprte ALO from these two gener: microscopic ppernce (Grm vribility), PYse, growth in 6.5% NCl, vncomycin susceptibility, rection with streptococcl group D ntigen, bile-esculin, nd growth t 45 C. Differentiting Leuconostoc spp. nd Lctobcillus spp. from ALO does not pose ny problem when the differences in microscopic ppernce nd biochemicl rections re considered. GC moles percent determintion of ALO strins suggests possible reltionship especilly to treptococcus spp., Pediococcus spp., nd A. viridns (Tble 5). ince there re both phenotypic similrities nd mjor differences between these gener, DNA-DNA hybridiztion investigtions re now being performed to clrify whether the isoltes represent seprte txon. ACKNOWLEDGMENT We re indebted to the stffs of the prticipting deprtments of clinicl microbiology t Frederiksberg Hospitl, igshospitlet, nd the Centrl Hospitls in Esbjerg, Hillerd, Nwstved, nd lgelse for supplying us with the isoltes nd to the clinicl deprtments t these hospitls for llowing us to study the ptient records. We thnk Lene Andressen nd Tine Nedergrd for excellent technicl ssistnce. EFEENCE 1. Csls, J. B., nd N. Pringler Dignostic tblets for bcteril identifiction, 3rd ed., p. 16. osco Dignostic, Tstrup, Denmrk. 2. Christensen, J. J., B. Korner, nd H. Kjergrd Aerococcus-like orgnism-n unnoticed urinry trct pthogen. APMI 97: Colmn, G Aerococcus-like orgnisms isolted from humn infections. J. Clin. Pthol. 2: Fcklm,., D. Hollis, nd M. D. Collins Identifiction of grm-positive coccl nd coccobcillry vncomycin-resistnt bcteri. J. Clin. Microbiol. 27: Jnosek, J., J. Eckert, nd A. Hudc Aerococcus viridns s custive gent of infectious endocrditis. J. Hyg. Epidemiol. Microbiol. Immunol. 1: Kern, W., nd E. Vnek Aerococcus bcteremi ssocited with grnulocytopeni. Eur. J. Clin. Microbiol. 6: Kilin, M., L. Mikkelsen, nd J. Henrichsen Txonomic study of viridns streptococci: description of treptococcus gordonii sp. nov. nd emended descriptions of treptococcus snguis (White nd Niven 1946), treptococcus orlis (Bridge nd neth 1982), nd treptococcus mitis (Andrewes nd Horder 196). Int. J. yst. Bct. 39: Lutrop, H., N. Hiby, A. Bremmelgrd, nd B. Korsger Bkteriologiske undersgelsesmetoder. FADL's Forlg, Copenhgen. 9. Nthvithrn, K. A.,. N. Arseculertne, H. A. Aponso,. VUertnm, L. Jysen, nd C. Nvrtmn Acute meningitis in erly childhood cused by Aerococcus viridns. Br. Med. J. 286: Prker, M. T., nd L. C. Bll treptococci nd erococci ssocited with systemic infection in mn. J. Med. Microbiol. 9: Downloded from on October 6, 218 by guest

5 VOL. 29, 1991 UINAY TACT INFECTION AND ALO Pien, F. D., W.. Wilson, K. Kunz, nd J. A. Wshington H Aerococcus viridns endocrditis. Myo Clin. Proc. 59: Pope, C. G., nd F. V. Linggood Purifiction of diphtheri toxoid. Br. J. Exp. Pthol. 2: ndstedt, K., J. Ursing, nd M. Wlder Thermotolernt Cmpylobcter with no or wek ctlse ctivity isolted from dogs. Curr. Microbiol. 8: neth, P. H. A., N. A. Mir, M. E. hrpe, nd J. G. Holt (ed.) Bergey's mnul of systemtic bcteriology, vol. 2. The Willims & Wilkins Co., Bltimore. 15. Untereker, W. J., nd B. A. Hnn Endocrditis nd osteomyelitis cused by Aerococcus viridns. Mt. ini J. Med. 43: Downloded from on October 6, 218 by guest