Laboratory Animals : opportunistic infections in laboratory rodents Klebsiella oxytoca
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Laboratory Animals : opportunistic infections in laboratory rodents Klebsiella oxytoca
Laboratory Animals http://lan.sagepub.com/ Klebsiella oxytoca: opportunistic infections in laboratory rodents Andre Bleich, Petra Kirsch, Hany Sahly, Jim Fahey, Anna Smoczek, Hans-Jürgen Hedrich and John P Sundberg Lab Anim 2008 42: 369 DOI: 10.1258/la.2007.06026e The online version of this article can be found at: http://lan.sagepub.com/content/42/3/369 Published by: http://www.sagepublications.com On behalf of: Laboratory Animals LtdLaboratory Animals Ltd Additional services and information for Laboratory Animals can be found at: Email Alerts: http://lan.sagepub.com/cgi/alerts Subscriptions: http://lan.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav >> Version of Record - Jul 1, 2008 What is This? Downloaded from lan.sagepub.com by guest on September 9, 2014 Klebsiella oxytoca: opportunistic infections in laboratory rodents Andre Bleich*, Petra Kirsch†, Hany Sahly‡, Jim Fahey§, Anna Smoczek*, Hans-Ju¨rgen Hedrich* and John P Sundberg§ *Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Carl- Neuberg-Str 1, 30625 Hannover, Germany; †Tierforschungszentrum der Universita¨t Ulm, Ulm, Germany; Institute for Infection Medicine, University Medical Center Schleswig Holstein Campus Kiel, Kiel, Germany; §The Jackson Laboratory, Bar Harbor, Maine, USA ‡ Summary Opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. This study was conducted to evaluate the role of Klebsiella oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytoca-induced lesions were studied from 2004 to early 2006 in naturally infected rodent colonies maintained at The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany. K. oxytoca infections were observed in substrains of C3H/HeJ mice, which carry the Tlr4Lps-d allele; in LEW.1AR1iddm rats, the latter being prone to diabetes mellitus; in immunodeficient NMRI-Foxn1nu mice; and in mole voles, Ellobius lutescens. The main lesions observed were severe suppurative otitis media, urogenital tract infections and pneumonia. Bacteriological examination revealed K. oxytoca as monocultures in all cases. Clonality analysis performed on strains isolated at the ZTM and TFZ (serotyping, pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction, sequencing of 16S rRNA and rpoB genes) revealed that the majority of bacteria belonged to two clones, one in each facility, expressing the capsule type K55 (ZTM) or K72 (TFZ). Two strains, one isolated at the ZTM and one at the TFZ, showed different PFGE and ERIC pattern than all other isolates and both expressed capsule type K35. In conclusion, K. oxytoca is an opportunistic pathogen capable of inducing pathological lesions in different rodent species. Keywords Diabetes mellitus; Klebsiella; otitis; pneumonia; TLR4; UGI Klebsiella spp. are opportunistic Gram-negative pathogens that can cause community-acquired severe pyogenic pneumonia in humans, with a high mortality rate if left untreated (Carpenter 1990, Prince et al. 1997, Ishida et al. 1998). The vast majority of Klebsiella spp. infections are associated with hospitalization, the urinary tract being the most common affected site. Klebsiella spp. infections in humans are mainly caused by Correspondence: A Bleich. Email: [email protected] Accepted 30 July 2007 # Laboratory Animals Ltd Klebsiella pneumoniae and to a lesser degree by K. oxytoca (Podschun & Ullmann 1998). Mice and rats experimentally infected with K. pneumoniae serve as models for a variety of diseases including pneumonia, endotoxaemia, sepsis, cystitis and pyolenephritis (Baker 1998). However, reports of naturally occurring infectious diseases in rodents due to Klebsiella spp., especially K. oxytoca, are very infrequent (Schneemilch 1976, Jackson et al. 1980, Boot & Walvoort 1986, Rao et al. 1987). Due to the elimination of many primary pathogens in experimental rodent colonies Downloaded from lan.sagepub.com by guest on September 9, 2014 DOI: 10.1258/la.2007.06026e. Laboratory Animals (2008) 42, 369 –375 370 A Bleich et al. over the last decades, the hygienic status of animals used in research has considerably improved. However, by maintaining animals with a well-defined hygienic status (specified pathogen free), opportunistic pathogens have become increasingly relevant as the causative agents of clinical disease and pathological lesions in laboratory animals. The aim of this study was to determine the role of K. oxytoca as an opportunistic pathogen in laboratory rodents. Therefore, K. oxytocainduced lesions were studied in naturally infected colonies of Mus musculus, Rattus norvegicus and Ellobius lutescens maintained in three different animal facilities, and K. oxytoca strains isolated from these rodents were characterized. Materials and methods formalin (TJL), processed routinely, embedded in paraffin, sectioned at 5 –6 mm and stained with haematoxylin and eosin. Depending on the lesion, organs or swabs were cultured on blood agar (at the TFZ: Mueller-Hinton agar with 5% sheep blood [Merck, Darmstadt, Germany]; at the ZTM: blood agar base no. 2 with 5% defibrinated sheep blood [Oxoid, Wesel, Germany]; at the TJL: Columbia agar with 5% defibrinated sheep blood [Northeast Labs, Waterville, ME, USA]) and Enterobacteriaceae-specific agar (Gassner [ZTM; Oxoid] or MacConkey’s agar [TFZ, TJL; Merck or Difco, Sparks, MD, USA, respectively]) for 24 h at 378C directly or after enrichment in broth medium (thioglycollate [TFZ, ZTM; Oxoid] or tryptose phosphate [TJL, Difco]) at 378C for up to one week. Study design From 2004 to early 2006, 92 cases of K. oxytoca infections from The Jackson Laboratory (TJL), Bar Harbor, USA, the Animal Research Centre (Tierforschungszentrum, TFZ) of the University of Ulm, Germany and the Central Animal Facility (ZTM) of the Hannover Medical School, Germany, were analysed for this study. Only animals displaying lesions, from which K. oxytoca was isolated as a monoculture, were included in this study. At the TFZ and ZTM, K. oxytoca isolated from cases as well as K. oxytoca isolated from non-affected animals during routinely performed hygienic monitoring programmes according to the Federation of European Laboratory Animal ScienceAssociatons(FELASA)recommendations (Nicklas et al. 2002) were assembled for further analyses. Typing of Klebsiella isolates Klebsiella isolates from clinical affected and unaffected animals were identified by the API20E system (bioMe´rieux, Marcy l’Etoile, France). Isolates from affected animals at the TFZ and ZTM as well as unaffected controls were also tested for assimilation of ethanolamine, histamine, D-melezitose and DL-3-hydroxybutyrate (Sigma-Aldrich, Munich, Germany) according to Monnet and Freney (1994) and subjected to serotyping and molecular analysis ( pulse field gel electrophoresis [PFGE], enterobacterial repetitive intergenic consensus polymerase chain reaction [ERIC-PCR], sequencing of 16S rRNA and rpoB genes). Serotyping and PFGE were performed at the Institute for Infection Medicine, Kiel, ERIC-PCR at the TFZ and sequencing was performed at the ZTM. Histological and bacteriological examination Clinically ill animals underwent necropsy in all three facilities. Animals were euthanized by CO2 asphyxiation. Swabs were taken from suppurative lesions and/or affected organs were removed aseptically and one part cultured as described below; the remaining samples were fixed in neutral buffered 4% formalin (TFZ, ZTM) or 10% acid –alcohol Serotyping, PFGE, ERIC-PCR and analysis of clonality To analyse clonality, strains were K-serotyped, subjected to PFGE and ERIC-PCR was performed. To determine the K-serotypes, the bacteria were grown on Worfel-Ferguson agar (0.025% w/v MgSO4, 0.1% w/v Ka2SO4, 0.2% w/v NaCl, 2% w/v saccharose [Merck], 0.2% w/v yeast extract and 1.5% Bacto agar [BD, Heidelberg, Laboratory Animals (2008) 42 Downloaded from lan.sagepub.com by guest on September 9, 2014 Klebsiella spp. in rodents 371 Germany]) for 24 h at 378C and for an additional 24 h at room temperature to promote capsular production. The K-serotypes of the isolates were determined by the capsular swelling method using K-specific antisera as described elsewhere (Podschun et al. 1993). PFGE patterns of the strains were determined after restriction of the bacterial DNA with the endonuclease XbaI as described previously (Sahly et al. 2000a). Strains that were indistinguishable, closely related or possibly related according to the Tenover’s criteria for the analysis of PFGE pattern (Tenover et al. 1995) and expressing identical K-serotype were regarded as clonal. K. oxytoca isolates were also typed by ERIC-PCR using the Ready-to-go RAPD Analysis Bead kit (Amersham Pharmacia, Little Chalfont, UK) and ERIC-1R primer 0 (5 -ATG-TAA-GCT-CCT-GGG-GAT-TCA0 C-3 ) (Granier et al. 2003). PCR was carried out with an initial denaturation step of 5 min at 958C, 45 amplification cycles of 1 min at 958C, 1 min at 368C, 2 min at 728C followed by a final extension step of 10 min at 728C. Sequencing Portions of 16S rRNA genes of Klebsiella isolates were amplified using primers A12 0 0 (5 -AAG-CCT-GAT-GCA-GCC-A-3 ) and A13 0 0 (5 -TTT-CGC-ACC-TGA-GCG-T-3 ) (Granier et al. 2003), the rpoB (RNA polymerase beta-subunit) encoding genes using primers 0 0 CM81b (5 -TGA-TCA-ACG-CCA-AGC-C-3 ) 0 and CM32b (5 -CGG-AAC-GGC-CTGACG-TTG-CAT-30 ) (Mollet et al. 1997). PCR was carried out using REDExtract-N-AMPPCR ReadyMix (Sigma-Aldrich) and annealing temperatures were set to 458C. Amplificates were loaded on a 2% SeaKem agarose gel (Biozym, Hessisch Oldendorf, Germany) containing SYBR Green (Gel Star, 4 mL/100 mL; Biozym), after 35 cycles of the PCR. PCR products were purified using the NucleoSpin Extract II kit (Macherey Nagel, Du¨ren, Germany) and sequenced. Sequence alignments were performed with the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990; www.ncbi.nlm.nih.gov/blast/Blast.cgi) and CLUSTAL W (Thompson et al. 1994; www.ebi.ac.uk/clustalw/). Sequences were submitted to GenBank (EF525558-EF525561). Results At TJL, K. oxytoca infections were observed in C3H/HeJ mice, which carry the Tlr4Lps-d allele (Poltorak et al. 1998). At the ZTM, infections were observed in C3H/HeJZtm mice (also carrying the Tlr4Lps-d allele) and in LEW.1AR1-iddm rats, the latter being prone to diabetes mellitus. Both strains were maintained in the same hygienic unit. In addition, lesions were observed in immunodeficient NMRI-Foxn1nu mice. In Ulm, K. oxytoca-induced lesions were detected in two mole voles, Ellobius lutescens. All cases are summarized in Table 1. Colony sizes of the affected breeding colonies were: an average of 20 voles at the TFZ, 60 – 70 C3H/HeJ mice and 70 – 80 LEW.1AR1-iddm rats at the ZTM. Gross necropsy and histology The main lesions observed were otitis media (that extended to cause osteolysis of the bulla in a vole), urogenital tract infections and pneumonia. Besides this, subcutaneous, intra-abdominal and liver abscesses, keratoconjunctivitis, Harderian gland adenitis, meningitis, infections of the oral cavity, maxilla and salivary glands were noted (Table 1, Figure 1). Lesions induced by K. oxytoca showed severe suppuration and extensive necrosis. Processes tended to spread to neighbouring tissue (Figure 1). Bacteriological examinations Klebsiella spp. were cultured from lesions described above, and biochemical identification (API20E and additional assimilation tests performed at the ZTM and TFZ) as well as sequencing of 16S rRNA and rpoB genes revealed K. oxytoca in all cases. Bacteria isolated from lesions, as well as K. oxytoca cultured from non-affected animals examined during routine health monitoring at the TFZ and the ZTM were further typed Downloaded from lan.sagepub.com by guest on September 9, 2014 Laboratory Animals (2008) 42 372 A Bleich et al. Table 1 Klebsiella oxytoca infections in laboratory rodents (Mus musculus, Rattus norvegicus, Ellobius lutescens) maintained at the Central Animal Facility, Hannover Medical School (ZTM), the Tierforschungszentrum, University of Ulm (TFZ) and The Jackson Laboratory, Bar Harbor (TJL) Cases Otitis UGI Species Strain Total ZTM M. musculus TFZ TJL R. norvegicus E. lutescens M. musculus C3H/HeJZtm NMRI-Foxn1nu LEW.1AR1-iddm 4 4 6 2 76 1 29 13 92 30 21 Total C3H/HeJ 2 Pneumonia Abscess† Other 2 1 3 19 8 1 16 21 9 20 6 Otitis media in mice; otitis media and osteolysis of the bulla in a vole † Subcutanous, intra-abdominal, intrahepatic Other: Keratoconjunctivitis and Harderian gland adenitis; meningitis; infection of the oral cavity, the maxilla, and salivary glands; lymphadenitis UGI: urogenital tract infection C3H/HeJZtm and C3H/HeJ mice carry the Tlr4Lps-d allele serologically and by molecular methods. Clonality analysis of the K. oxytoca strains revealed two clones, one in each facility, with two exceptions. At the end of this study, one strain was isolated from a keratoconjunctivitis in an NMRI-Foxn1nu mouse at the ZTM and one strain was isolated from a parotid gland abscess of a vole at the TFZ. Both strains showed different PFGE and ERIC patterns than all other isolates from each facility. Both expressed the capsular type K35. The clonally indistinguishable strains isolated at the ZTM expressed the capsule type K55 and showed identical PFGE and ERIC-PCR patterns. In contrast, the clonally identical strains isolated at the TFZ showed different PFGE and ERIC-PCR pattern than the ZTM isolates, but were indistinguishable among themselves, and expressed the serotype K72. Sequencing revealed identical 16S rRNA and rpoB gene sequences in all indistinguishable ZTM strains investigated. However, ZTM and TFZ strains showed differences in both gene sequences. Discussion In human and veterinary medicine, Klebsiella spp. infections are primarily caused by K. pneumoniae. This applies also for laboratory rabbits and rodents. Here, we describe the identification of K. oxytoca in lesions of three different rodent species. In all lesions described, K. oxytoca was obtained as Laboratory Animals (2008) 42 a monoculture, strongly suggesting its causative role. Besides the two infections in Ellobius spp. that are not known to have any immune defect, K. oxytoca-induced lesions were restricted to mice and rats displaying characteristic strain-specific features. The two C3H/HeJ substrains are bacterial lipopolysaccharide (LPS) hyporesponsive due to a defect in their toll-like receptor 4 (TLR4) protein (Poltorak et al. 1998). Defective alleles of this receptor are associated with increased susceptibility to Gram-negative infections in humans (Agnese et al. 2002) and mice (Bernheiden et al. 2001, Branger et al. 2004). NMRI-Foxn1nu mice lack a thymus (T-lymphocytes) and are therefore immunodeficient. In addition, these athymic nude mice lack functional eyelashes due to severe follicular dystrophy and might be more susceptible to keratoconjunctivitis (which was seen in these mice) than other mouse strains (Bazille et al. 2001). LEW.1AR1-iddm rats develop diabetes mellitus (Lenzen et al. 2001), a risk factor for Klebsiella spp. induced urogenital tract infection (Chan et al. 1993) and liver abscesses (Lee et al. 2001) in humans. In addition to predisposing conditions of the host, antibiotic treatment and virulence factors of the bacterium play a role in the pathogenesis of Klebsiella spp. induced lesions. Abnormal colonization with resistant K. pneumoniae has been described after antibiotic treatment of nude rats and mice (Hansen 1995), and antibiotic treatment is Downloaded from lan.sagepub.com by guest on September 9, 2014 Klebsiella spp. in rodents 373 Figure 1 Klebsiella oxytoca-induced lesions. (A,B) Macroscopic examples of K. oxytoca-induced lesions. Osteolysis of the bulla in a vole (A) and urogenital tract infection in a LEW.1AR1-iddm rat (B) are shown. The forceps in (B) identifies the mesorchium and testicle. (C,D) Massive exudation and suppuration in the middle ear of a C3H/HeJ mouse resulting in severe acute suppurative otitis media. Bar ¼ 200 mm (C), 50 mm (D). (E,F) Direct scan (E) and magnification (F) of a lung from a C3H/HeJ. Note the severe fibrinopurulent necrotizing lobar pneumonia in the upper lobe (E). Higher magnification (F) illustrates the massive infiltration of polymorphonuclear lymphocytes and uniform mass of bacilli that were shown by culture to be K. oxytoca. Bar ¼ 2 mm (E), 100 mm (F). (G–J) A C3H/HeJ mouse with severe acute fibrinopurulent pneumonia (G,I,J) that extends to the heart to cause pericarditis (G,H). Bar ¼ 200 mm (G), 100 mm (H,I,J). (K,L) Suppurative meningitis of a C3H/HeJ mouse with systemic K. oxytoca infection. Note the inflammatory cells in the meninges (K, top) extending deep into the dorsal median sulcus (boxed area). The boxed area in (L) consists of numerous polymorphonuclear leukocytes and a uniform population of bacilli. Bar ¼ 200 mm (K), 100 mm (L). (M –P) Pyelonephritis caused by an ascending K. oxytoca infection in a C3H/HeJ mouse. Direct scan of the affected kidney (M). Higher magnification shows areas of extensive necrosis in the pelvis and massive neutrophilic infiltration within and around the necrotic tubules (N,O,P). Bar ¼ 1 mm (M), 200 mm (N), 100 mm (O,P) Downloaded from lan.sagepub.com by guest on September 9, 2014 Laboratory Animals (2008) 42 374 A Bleich et al. associated with increased colonization with Klebsiella spp. in humans as well. In the infected animal rooms at the ZTM, antibiotic treatment was performed with tetracycline (tetracycline hydrochloride 0.7 g/L drinking water for two weeks; bela-pharm, Vechta, Germany) at the end of 2003; however, K. oxytoca was also isolated from clinically ill animals before antibiotic treatment was initiated. However, prior to the use of antibiotics, lesions compatible with infection of K. oxytoca were never observed. Klebsiella spp. are capable of producing a prominent capsule composed of complex acidic polysaccharides, which are likely major determinants of pathogenicity, at least for K. pneumoniae (Podschun & Ullmann 1998). Based on the structural variability of the capsular polysaccharides, Klebsiella spp. has been classified into 77 serotypes which differ in their pathogenicity and epidemiological relevance (Sahly et al. 2000b). The majority of K. oxytoca isolated from lesions at the ZTM were classified as the K55 serotype. K. oxytoca of the same capsule type was identified as the causative agent of septicaemia in human neonatal wards, suggesting a possible enhanced virulence of this serotype (Morgan et al. 1984, Tullus et al. 1992). In conclusion, this study describes K. oxytoca as an opportunistic pathogen capable of inducing pathological lesions in three different rodent species. Interestingly, the appearance of cases in TLR4-deficient, diabetic prone or immunodeficient animals and the observation that antibiotic treatment is likely a predisposing factor for abnormal colonization and for induction of lesions show parallels to the situation in human medicine. Acknowledgements This work was supported in part by grants from the National Institutes of Health (RR00173 to JPS, CA34196 to The Jackson Laboratory for core facility support) and from the DFG (SFB621, HJH). The authors thank C Elvers and I Ko¨hn for their technical assistance. References Agnese DM, Calvano JE, Hahm SJ, et al. (2002) Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an Laboratory Animals (2008) 42 increased risk of Gram-negative infections. Journal of Infectious Diseases 186, 1522–5 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403– 10 Baker DG (1998) Natural pathogens of laboratory mice, rats, and rabbits and their effects on research. Clinical Microbiology Reviews 11, 231–66 Bazille PG, Walden SD, Koniar BL, Gunther R (2001) Commercial cotton nesting material as a predisposing factor for conjunctivitis in athymic nude mice. Lab Animal (NY) 30, 40– 2 Bernheiden M, Heinrich JM, Minigo G, et al. (2001) LBP, CD14, TLR4 and the murine innate immune response to a peritoneal Salmonella infection. Journal of Endotoxin Research 7, 447–50 Boot R, Walvoort HC (1986) Opportunistic infections in hysterectomy-derived, barrier-maintained guinea pigs. Laboratory Animals 20, 51– 6 Branger J, Knapp S, Weijer S, et al. (2004) Role of toll-like receptor 4 in Gram-positive and Gram-negative pneumonia in mice. Infection and Immunity 72, 788–94 Carpenter JL (1990) Klebsiella pulmonary infections: occurrence at one medical center and review. Reviews of Infectious Disease 12, 672– 82 Chan RK, Lye WC, Lee EJ, Kumarasinghe G (1993) Nosocomial urinary tract infection: a microbiological study. Annals of the Academy of Medicine, Singapore 22, 873–7 Granier SA, Plaisance L, Leflon-Guibout V, et al. (2003) Recognition of two genetic groups in the Klebsiella oxytoca taxon on the basis of chromosomal beta-lactamase and housekeeping gene sequences as well as ERIC-1 R PCR typing. International Journal of Systematic and Evolutionary Microbiology 53, 661– 8 Hansen AK (1995) Antibiotic treatment of nude rats and its impact on the aerobic bacterial flora. Laboratory Animals 29, 37 –44 Ishida T, Hashimoto T, Arita M, Ito I, Osawa M (1998) Etiology of community-acquired pneumonia in hospitalized patients: a 3-year prospective study in Japan. Chest 114, 1588–93 Jackson NN, Wall HG, Miller CA, Rogul M (1980) Naturally acquired infections of Klebsiella pneumoniae in Wistar rats. Laboratory Animals 14, 357– 61 Lee KT, Wong SR, Sheen PC (2001) Pyogenic liver abscess: an audit of 10 years’ experience and analysis of risk factors. Digestive Surgery 18, 459– 65, Discussion 465 –6 Lenzen S, Tiedge M, Elsner M, et al. (2001) The LEW.1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus. Diabetologia 44, 1189–96 Mollet C, Drancourt M, Raoult D (1997) rpoB sequence analysis as a novel basis for bacterial identification. Molecular Microbiology 26, 1005–11 Downloaded from lan.sagepub.com by guest on September 9, 2014 Klebsiella spp. in rodents 375 Monnet D, Freney J (1994) Method for differentiating Klebsiella planticola and Klebsiella terrigena from other Klebsiella species. Journal of Clinical Microbiology 32, 1121–2 Morgan ME, Hart CA, Cooke RW (1984) Klebsiella infection in a neonatal intensive care unit: role of bacteriological surveillance. Journal of Hospital Infection 5, 377–85 Nicklas W, Baneux P, Boot R, et al. (2002) Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Laboratory Animals 36, 20 –42 Podschun R, Ullmann U (1998) Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clinical Microbiology Reviews 11, 589–603 Podschun R, Sievers D, Fischer A, Ullmann U (1993) Serotypes, hemagglutinins, siderophore synthesis, and serum resistance of Klebsiella isolates causing human urinary tract infections. Journal of Infectious Diseases 168, 1415–21 Poltorak A, He X, Smirnova I, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–8 Prince SE, Dominger KA, Cunha BA, Klein NC (1997) Klebsiella pneumoniae pneumonia. Heart & Lung: The Journal of Critical Care 26, 413–17 Rao GN, Hickman RL, Seilkop SK, Boorman GA (1987) Utero-ovarian infection in aged B6C3F1 mice. Laboratory Animal Science 37, 153– 8 Sahly H, Podschun R, Oelschlaeger TA, et al. (2000a) Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae. Infection and Immunity 68, 6744– 9 Sahly H, Podschun R, Ullmann U (2000b) Klebsiella infections in the immunocompromised host. Advances in Experimental Medicine and Biology 479, 237–49 Schneemilch HD (1976) A naturally acquired infection of laboratory mice with Klebsiella capsule type 6. Laboratory Animals 10, 305– 10 Tenover FC, Arbeit RD, Goering RV, et al. (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–80 Tullus K, Ayling-Smith B, Kuhn I, Rabsch W, Reissbrodt R, Burman LG (1992) Nationwide spread of Klebsiella oxytoca K55 in Swedish neonatal special care wards. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica 100, 1008– 14 Downloaded from lan.sagepub.com by guest on September 9, 2014 Laboratory Animals (2008) 42