J. Med. Microbiol. Ð Vol. 50 $2001), 396±406 # 2001 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 ANTIMICROBIAL SUSCEPTIBILITY Natural antibiotic susceptibility of Klebsiella pneumoniae, K. oxytoca, K. planticola, K. ornithinolytica and K. terrigena strains INGO STOCK and BERND WIEDEMANN Institut fuÈ r Medizinische Mikrobiologie und Immunologie, Pharmazeutische Mikrobiologie, UniversitaÈt Bonn, Germany The natural susceptibility of 221 Klebsiella strains to 71 antibiotics was examined. The strains were isolated from clinical specimens and the environment, and belonged to K. pneumoniae subsp. pneumoniae n 40), K. pneumoniae subsp. ozaenae 37), K. pneumoniae subsp. rhinoscleromatis 10), K. oxytoca 44), K. planticola 40), K. ornithinolytica 25)and K. terrigena 25). MIC values were determined by a microdilution procedure in IsoSensitest broth according to the German standard DIN). All Klebsiella spp. were naturally resistant or intermediate to amoxicillin, ticarcillin and to antibiotics to which other Enterobacteriaceae are also intrinsically resistant. Klebsiella spp. were naturally sensitive or intermediate to several penicillins, all tested cephalosporins, aminoglycosides, quinolones, tetracyclines, trimethoprim, co- trimoxazole, chloramphenicol and nitrofurantoin. K. pneumoniae subsp. ozaenae and subsp. rhinoscleromatis strains were generally more susceptible to antibiotics than strains of other Klebsiella taxa. K. pneumoniae subsp. rhinoscleromatis was the most susceptible taxon, being highly susceptible to cefuroxime, anti-folates and naturally intermediate to erythromycin and clarithromycin. K. pneumoniae subsp. ozaenae was most susceptible to glycopeptides. K. oxytoca and K. terrigena strains were least susceptible to cefazoline, cefoperazone and fosfomycin, respectively. The results of the present study describe a database of the natural antimicrobial susceptibility of Klebsiella spp., which can be used for the validation of antibiotic susceptibility results of these bacteria. MIC patterns to â-lactams indicate the expression of chromosomally encoded class A â-lactamases in all the species, including the subspecies of K. pneumoniae. Similar natural susceptibility patterns of K. planticola and K. ornithinolytica to all tested antibiotics support the status of K. ornithinolytica as a biovar of K. planticola. Introduction pneumoniae subsp. ozaenae $K. OzaenaeA) and K. pneumoniae subsp. rhinoscleromatis $K. Rhino- Klebsiella spp. are opportunist pathogens that cause a scleromatisA) are restricted to certain body sites and in wide range of infections in man. They account for 8% most cases affect only the human nose. K. Ozaenae is of all nosocomial bacterial infections in the USA and the cause of an atrophic rhinitis called ozena, but in Europe [1]. Most frequently, Klebsiella spp. are sporadic cases of other infections due to this organism isolated as causative agents of urinary tract infections, are known [3, 4]. K. Rhinoscleromatis is the aetiolo- pneumonia, bacteraemia, neonatal sepsis and wound gical agent of rhinoscleroma, a chronic granulomatous infections [1]. The leading organism in these infections infection of the nose, which is endemic in several is K. pneumoniae subsp. pneumoniae $K. Pneu- countries [5]. While originally considered to be without moniaeA) [2], followed by K. oxytoca. In contrast to clinical signi®cance and restricted to water, botanical diseases caused by these taxa, infections due to K. and soil environments, K. planticola and K. terrigena have been shown to occur in clinical specimens [5± 10]. K. planticola has been isolated from human Received 25 Feb. 2000; revised version received 16 Oct. infections with a frequency of 3.5±20% among clinical 2000; accepted 25 Oct. 2000. Corresponding author: Dr I. Stock $e-mail: ingostock@ hotmail.com). Ataxonomic style according to Le Minor [2]. NATURAL ANTIBIOTIC SUSCEPTIBILITY OF KLEBSIELLA SPP. 397 isolates of Klebsiella spp. [6±9]. Human strains have Ehrlich Society conducted in 1986 at 30 centres in been isolated mainly from respiratory tract secretions, Germany, Switzerland and Austria. Twenty two K. wounds and urine [9]. Recently, K. planticola was also Ozaenae and some further strains ± K. Rhinoscler- isolated from newborns in a neonatal ward [11]. It is omatis $n 6), K. ornithinolytica $2), K. terrigena $2) likely that K. planticola causes human disease, because and K. planticola $1) ± were kindly provided by G. isolates have been recovered from monomicrobial Stempfel and H. Grimm $Weingarten, Germany). Apart specimens and could have been assigned to the from K. Rhinoscleromatis strains, which were gathered corresponding infections [9]. K. planticola has the in the last decade, these strains were collected during ability to express putative virulence factors similar to 1996±1998 and originated from different hospitals and K. Pneumoniae, such as type 1 and type 3 ®mbriae from outpatients in cities in southern Germany. Most of [12], and possesses the high-pathogenicity island of the remaining K. Ozaenae, four K. Rhinoscleromatis, many virulent Yersinia strains [13]. In contrast to K. 20 K. planticola,20K. terrigena and 23 K. planticola, K. terrigena strains seem to be rarely ornithinolytica strains were kindly provided by R. associated with human infections. Podschun and Podschun $Kiel, Germany); these strains served as Ullmann found these bacteria in 0.4% of clinical reference strains. They had also been identi®ed and Klebsiella isolates [10]. However, because most serotyped at the Hygiene-Institut of Kiel and were of commercial identi®cation systems do not permit a clinical or environmental origin. K. terrigena ATCC reliable identi®cation of K. terrigena and K. planticola, 33257, K. terrigena ATCC 33629, K. terrigena ATCC the true clinical signi®cance of these Klebsiella species 33630, K. planticola ATCC 33558, K. planticola is unknown. In 1989, Sakazaki et al. proposed the CUETM78117 and six further K. planticola strains name `K. ornithinolytica' for ornithine decarboxylase- were from the culture collection of Merlin-Diagnostika and indole-positive Klebsiella strains [14]. This name $Bornheim, Germany). No clinical isolates were repeat is well accepted in Japan but not in the USA. The isolations from a given patient or patients on the same distinctness of K. ornithinolytica from K. planticola ward. needs to be con®rmed, as DNA±DNA relatedness studies in the USA and Japan gave different results Identi®cation [14, 15]. The clinical signi®cance of K. ornithinolytica remains obscure, even though clinical isolates of this All strains were identi®ed to the genus level with a species are not uncommon $unpublished data). Finally, commercial identi®cation system for Enterobacteria- in 1999 a sixth Klebsiella species was created. Based ceae $Micronaut-E, Merlin-Diagnostika). This identi®- on 16S rRNA genes sequences, it was found that cation system includes biochemical key reactions for Calymmatobacterium granulomatis, the aetiological Enterobacteriaceae species with clinical signi®cance. agent of a chronic granulomatous genital infection The inoculum for the identi®cation tests was prepared called donovanosis, is highly related to Klebsiella spp. from overnight cultures on solid media in physiological [16], implying its reclassi®cation as K. granulomatis saline and was c.106 cfu=ml. The incubation times [17]. were 24 h, the incubation temperature was 368C $Æ 18C). To identify klebsiellae to species level, the Despite their occurrence in clinical specimens, there is Micronaut-E system and additional assimilation tests little information about the antibiotic susceptibility and $reactions according to Podschun and Ullmann [1] and no information about the natural antibiotic sensitivity Monnet and Freney [18]), i.e., utilisation of ethanola- and resistance of Klebsiella strains which do not mine $EA), histamine $HA), D-melezitose $MZ) and m- belong to K. Pneumoniae and K. oxytoca. Data about hydroxybenzoate $HB) $all chemicals obtained from the natural antibiotic susceptibility of K. Pneumoniae Sigma, Deisenhofen, Germany) were performed in and K. oxytoca are also rare. The aim of the present microtitration plates. In each plate assimilation patterns study was to create a database of the natural of four Klebsiella strains were tested in triplicate. susceptibility of all known Klebsiella species and Aqueous solutions of carbon source 10 g=L $EA, HA subspecies, except K. granulomatis, to a wide range and HB) and carbon source 20 g=L $MZ and glucose ± of antibiotics. The data from 221 Klebsiella strains growth control) were prepared, sterilised by ®ltration tested with 71 antibiotics could be valuable for the and stored at 48C. Lines A±D and line G of the validation of routine susceptibility test results and their microtitration plate were given 25 ìl of the appropriate consistency with identi®cation to species or subspecies carbon sources. Sterilised water was added to lines E, F level. and H, which served as negative controls. Then 100 ìl of AUX-Medium $bioMerieux, Marcy l'Etoile, France) were added to each well of the test plate. Finally, 50 ìl Materials and methods of the bacterial suspensions were added to the wells in Bacterial strains lines A±G, and 50 ìl of physiological saline were added to the wells in line H. Overnight cultures of the A total of 221 Klebsiella strains was examined. The bacteria grown on solid medium $IsoSensitest Agar, vast majority of the K. Pneumoniae and K. oxytoca Oxoid) at 368C were used for the preparation