Laboratory Animals : opportunistic infections in laboratory rodents Klebsiella oxytoca

Laboratory
Animals
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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
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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: bleich.andre@mh-hannover.de
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
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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
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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
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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
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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)
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Laboratory Animals (2008) 42
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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.
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