Cytotoxic and Pathogenic Properties of Klebsiella
oxytoca Isolated from Laboratory Animals
Alison Darby1., Kvin Lertpiriyapong1.¤, Ujjal Sarkar4, Uthpala Seneviratne4, Danny S. Park2,
Eric R. Gamazon3, Chara Batchelder1, Cheryl Cheung1, Ellen M. Buckley1, Nancy S. Taylor1, Zeli Shen1,
Steven R. Tannenbaum4,5, John S. Wishnok4, James G. Fox1,4*
1 Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 2 Pharmaceutical Sciences and
Pharmacogenomics Graduate Program, University of California, San Francisco, California, United States of America, 3 Section of Genetic Medicine, Department of
Medicine, The University of Chicago, Chicago, Illinois, United States of America, 4 Department of Biological Engineering, Massachusetts Institute of Technology,
Cambridge. Massachusetts, United States of America, 5 Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of
America
Abstract
Klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. Studies
suggest that in humans K. oxytoca exerts its pathogenicity in part through a cytotoxin. However, cytotoxin production in
animal isolates of K. oxytoca and its pathogenic properties have not been characterized. Furthermore, neither the identity of
the toxin nor a complete repertoire of genes involved in K. oxytoca pathogenesis have been fully elucidated. Here, we
showed that several animal isolates of K. oxytoca, including the clinical isolates, produced secreted products in bacterial
culture supernatant that display cytotoxicity on HEp-2 and HeLa cells, indicating the ability to produce cytotoxin. Cytotoxin
production appears to be regulated by the environment, and soy based product was found to have a strong toxin induction
property. The toxin was identified, by liquid chromatography-mass spectrometry and NMR spectroscopy, as low molecular
weight heat labile benzodiazepine, tilivalline, previously shown to cause cytotoxicity in several cell lines, including mouse
L1210 leukemic cells. Genome sequencing and analyses of a cytotoxin positive K. oxytoca strain isolated from an abscess of a
mouse, identified genes previously shown to promote pathogenesis in other enteric bacterial pathogens including ecotin,
several genes encoding for type IV and type VI secretion systems, and proteins that show sequence similarity to known
bacterial toxins including cholera toxin. To our knowledge, these results demonstrate for the first time, that animal isolates
of K. oxytoca, produces a cytotoxin, and that cytotoxin production is under strict environmental regulation. We also
confirmed tilivalline as the cytotoxin present in animal K. oxytoca strains. These findings, along with the discovery of a
repertoire of genes with virulence potential, provide important insights into the pathogenesis of K. oxytoca. As a novel
diagnostic tool, tilivalline may serve as a biomarker for K oxytoca-induced cytotoxicity in humans and animals through
detection in various samples from food to diseased samples using LC-MS/MS. Induction of K. oxytoca cytotoxin by
consumption of soy may be in part involved in the pathogenesis of gastrointestinal disease.
Citation: Darby A, Lertpiriyapong K, Sarkar U, Seneviratne U, Park DS, et al. (2014) Cytotoxic and Pathogenic Properties of Klebsiella oxytoca Isolated from
Laboratory Animals. PLoS ONE 9(7): e100542. doi:10.1371/journal.pone.0100542
Editor: Stefan Bereswill, Charite?-University Medicine Berlin, Germany
Received January 31, 2014; Accepted May 28, 2014; Published July 24, 2014
Copyright:  2014 Darby et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by NIH grants T32-RR007036 (J.G.F.) and P30-ES002109 (MIT Center for Environmental Health Sciences). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: jgfox@mit.edu
. These authors contributed equally to this work.
¤ Current address: East Carolina University, Brody School of Medicine, Department of Comparative Medicine, Greenville, North Carolina, United States of America
Introduction patients, including children and neonates [3,4,5]. K. oxytoca also is
purported to be an etiological agent of antibiotic-associated
Klebsiella oxytoca is a non-motile, gram-negative rod-shaped hemorrhagic colitis (AAHC) in adults and adolescents. Stool
bacterium belonging to the family Enterobacteriaceae. K. oxytoca is cultures of individuals with AAHC contain K. oxytoca, but do not
ubiquitous in the environment [1] and can be cultured from the contain organisms associated with diarrhea, such as Clostridium
skin, mucous membranes, oropharynx and intestines of healthy difficle, Campylobacter spp., Salmonella spp., Yersinia spp.,
humans and animals, as well as a variety of tissues from clinically Shigella spp., and E. coli O157 [6]. AAHC patients develop
affected humans and animals [2]. clinical signs following antibiotic and/or anti-inflammatory
In humans, K. oxytoca can be cultured from the stool of 8–10% therapy; these typically include bloody diarrhea, severe abdominal
of healthy adults [3]. Although most K. oxytoca-infected individ- cramping, and segmental hemorrhagic colitis as visualized by
uals remain asymptomatic, K. oxytoca is considered an opportu- colonoscopy, most commonly in the ascending colon and the
nistic pathogen and is now recognized as a clinically significant cecum [4,7,8]. Clinical signs are self-limiting and resolve several
pathogen associated with nosocomial infections in hospitalized days after antibiotics are discontinued. K. oxytoca has been
PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e100542
Klebsiella oxytoca in Laboratory Animals
cultured from human patients with septicemia, bacteremia, septic
arthritis, soft tissue infections, cholecystitis, urinary tract infections,
and more recently from colicky neonates [1,9,10,11,12,13,14,15].
Intestinal overgrowth of K. oxytoca has also been observed among
children with celiac disease [16].
In animals, K. oxytoca has been isolated from apparently healthy
sentinel rodents being monitored for pathogens in health
surveillance programs and from utero-ovarian infections including
suppurative endometritis, salpingitis, perioophoritis and peritonitis
in aged B6C3F1 mice on a long term carcinogenicity study
[17,18]. K. oxytoca was also cultured from cases of suppurative
otitis media, urogenital tract infections and pneumonia in C3H/
HeJ and NMRI-Foxn1 (nu) mice, LWE.1AR1 rats, and in mole
voles [19]. Additionally, K. oxytoca was recently cultured from
three breeding colonies of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
(NSG) mice with chronic renal inflammation and ascending
Figure 1. Tilivalline structure, chemical formula, and mass.
urinary tract infections [20]. An outbreak of K. oxytoca entero- doi:10.1371/journal.pone.0100542.g001
colitis on a rabbit farm was also reported [21]. In 2006,
Hogenauer et al. developed a model of AAHC by administering
strains of K. oxytoca, and correspondingly absent in negative
amoxicillin-clavulanate followed by orally infecting rats with a
strains under identical growth conditions, and ascertain whether
strain of K. oxytoca cultured from a patient with AAHC. The
tilivalline induced cell abnormalities and death in a cytotoxicity
Sprague-Dawley rats developed intestinal lesions primarily in the
cecum. K. oxytoca assay.was subsequently cultured from the rats with
enterocolitis, fulfilling Koch’s postulates and establishing the
positive association between AAHC and K. oxytoca infection [7]. Materials and Methods
The ability of the human strain of K. oxytoca to colonize and cause
Strains of K. oxytoca
disease in rats experimentally [7] also emphasizes the cross-species
Sixty four isolates of K. oxytoca isolated from various laboratory
infecting capability of K. oxytoca. Although the experimental
animal species were acquired from several sources and used in this
infection study suggests that human strains of K. oxytoca can infect
study. Of the 64 isolates, 48 were from mice (obtained from
rats, no evidence to date suggests that K. oxytoca strains from rats
Charles River Laboratories (CRL), Jackson Laboratory, MIT
or from other animal species can naturally infect humans and vice
Comparative Medicine diagnostic lab, and National Institute of
versa.
Environmental Sciences); 4 isolates were from rats (CRL), 8 from
K. oxytoca strains associated with colitis and mucocutaneous
non-human primates (CRL), 3 from pigs (CRL), and 1 from a
infections in humans can produce a cytotoxin, which may, in part,
guinea pig (CRL). In addition, a non-toxigenic K. oxytoca strain,
explain K. oxytoca pathogenesis [7]. The cytotoxin described in
ATCC 13182 [27], was obtained from American Type Culture
1989 and 1992 caused cell rounding and cell death when applied
in vitro Collection (ATCC) and used as a control.to various cell lines including HEp-2, HeLa, CHO, and
Vero cells [22,23]. However, cytotoxin production in K. oxytoca
isolated from animals has not been described [18,19,20]. Verification of K. oxytoca isolates
Furthermore, despite the known clinical effects of K. oxytoca in The identities of all 64 strains, previously identified by outside
humans and animals and the discovery of cytotoxin production in sources as K. oxytoca, were verified by a combination of API 20E
this bacterium, a complete portrait of its pathogenic mechanisms is test (bioMerieux, Marcy l’Etoile, France), polygalacturonase (pehX)
lacking. We hypothesized that - similar to K. oxytoca strains in gene amplification, and 16S rRNA gene sequencing. K. oxytoca
humans - K. oxytoca isolates of animal origin also produce DNA was used for subsequent molecular analyses with the Roche
cytotoxin and that K. oxytoca contain a repertoire of genes that High Pure PCR Template Kit according to the manufacturer’s
promote pathogenicity. To test this hypothesis, we obtained and recommendations (Roche Applied Science, Indianapolis, IN). For
characterized antibiotic resistance patterns in K. oxytoca strains pehX gene amplification, forward primer PEH C (5’ GAT ACG
from various animal species and investigated their ability to GAG TAT GCC TTT ACG GTG -3’) and reverse primer PEH
produce cytotoxin under various growth conditions using standard D (5’- TAG CCT TTA TCA AGC GGA TAC TTG -3’)
in vitro cytotoxicity assays previously described [22,23]. Further- (Integrated DNA Technologies, San Diego, CA) [28] were used.
more, we sequenced the genome of a cytotoxin-positive, clinical PCR amplification was performed using PuReTaq Ready-To-Go
isolate from diseased tissue of a mouse and performed in silico PCR Beads (GE Healthcare, Piscataway, NJ) as previously
analysis to gain insight into its potential pathogenic properties. described [28]. The PCR products were resolved on 2% agarose
Some of the natural exotoxins produced by Streptomyces and gel by electrophoresis and stained with ethidium bromide before
Micrococci species have a common pyrrolo-benzo-diazepine (PBD) visualization. Primer 9F (positions 9 to 27 in the forward direction)
moiety [24,25]. These area typically cytotoxic class of compounds and primer 1541 (positions 1525 to 1541 in the reverse direction)
consisting of mainly three rings [24]. Tilivalline (Figure 1), for were used to amplify the 16S rRNA genes from K. oxytoca as
example, is cytotoxic to mouse leukemia L1210 cells [25,26]. previously described [29]. The PCR products were then purified
However, the molecular mechanisms of cytotoxicity induced by using the QIAquick PCR purification kit (Qiagen, Valencia, CA)
these compounds are a matter of active investigation. With the and sequenced with an ABI Prism cycle sequencing kit (BigDye
previous results in mind, we suspected that the cytotoxic activity Terminator cycle sequencing kit) on an ABI 3500 genetic analyzer
present in the supernatant of laboratory animal strains of K. (Applied Biosystems, Foster City, CA). Sequences were compared
oxytoca might also be tilivalline. We therefore set out to determine directly with the National Center for Biotechnology Information
if tilivalline was present in the culture broth of cytotoxic-positive (NCBI) Genbank nucleotide database by BLAST search.
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Klebsiella oxytoca in Laboratory Animals
Antimicrobial Susceptibility bacteria liquid culture was centrifuged at 8,0006g for 5 minutes.
Antimicrobial susceptibility testing was performed using the disk The resulting supernatant was then filtered through 0.2 mm
diffusion method according to the standards set by Clinical and Acrodisc syringe filters (PALL Corporation, Port Washington, NY)
Laboratory Standards Institute. Briefly, tryptic soy broth (TSB) to exclude bacteria. Supernatants were either used immediately or
(BD, Franklin Lakes, NJ) was inoculated with K. oxytoca from 24- stored at 220uC until assayed. After 48 hours of incubation in
hour blood agar cultures and incubated at 37uC for 1 hr. The 37uC incubator aerated with 5% CO2, the percentage of cells
cultures were spread evenly on Meuller Hinton agar and antibiotic adhering to the bottom of the well of 24-well plates (% confluency)
discs were aseptically placed on the agar surface. The following indicative of live cells was assessed using an Olympus CK2
antibiotic discs were used: ampicillin 10mg, cephalothin 30mg, inverted phase contrast microscope at 4, 10 and 306 magnifica-
amoxicillin/clavulanic acid 20/10mg, trimethoprim/sulfamethox- tion. To facilitate visualization of the cells and to determine the
azole 1.25/23.75mg, enrofloxacin 5mg, and gentamicin 10mg total area of cells adhered to the well, Diff Quick straining was also
(Remel, Lexington, KS). Samples were incubated aerobically for performed. Cytotoxin-positive samples were characterized as ,
18–24 hours at 37uC and the zones of inhibition were measured to 50% confluency i.e., greater than 50% cell rounding and
the nearest millimeter. Measurements were categorized as detachment, as compared to negative control samples (TSB media
susceptible, intermediate susceptibility, or resistant based on the only or supernatant of a non-toxigenic K.oxytca strain, ATCC
established values for Klebsiella spp. reported by the manufacturer 13182) [27]. Negative control samples had a monolayer of cells
(BBL, Becton, Dickinson and Company, Sparks, MD). with minimal cell rounding or detachment and confluency in the
range of 80% to nearly 100%. The cytotoxicity assay was
Bacterial Cultures performed at least three times in duplicate wells for each K.
To propagate K. oxytoca, frozen stocks of bacteria were seeded oxytoca to confirm cytotoxin production status.
on blood agar (Remel) and grown for 24 hours at 37uC. To
investigate the effects of environmental conditions on cytotoxin Heat Inactivation and proteases, trypsin, DNase, and
production, a single colony of K. oxytoca from a 24-hour culture RNase treatment of Cytotoxin
was inoculated into 8 ml of tryptic soy broth (TSB) and then A 24-hour culture of K. oxytoca, 09-7231, was inoculated into
subjected to one of four conditions of variable oxygen content. TSB liquid media and grown under microaerobic conditions with
The first condition was a previously established growth condition gentle agitation for 16 hours. One milliliter of filtered supernatant
used in investigating cytotoxin production in human isolates of K. was obtained and subjected to heat treatment by boiling in a test
oxytoca [22,23]. This involved culturing K. oxytoca in a 37uC tube for 20 minutes or treated with protease (1 mg/ml of
incubator without agitation for 16 hours with 5% CO2. The supernatant) or trypsin (0.5 units/100 ml) at 37 uC with continuous
second method was similar to the first method except CO2 was not shaking (70 rpm) for 2 hours. For protease and trypsin treatments,
provided and gentle agitation at 100 rpm was implemented The the treated supernatant was subjected to centrifugation at 3000 g
third condition was microaerobic condition (10% CO2, 10% H2, for 1 minute to remove protease and trypsin agarose. Protease and
80% N2) in GasPak (BD, Franklin Lakes, NJ) jars, as previously trypsin agarose (Sigma, St Louis, MO) was reconstituted according
described [30]; the culture was incubated at 37uC with gentle to manufacturer’s recommendations. DNase (20 units/ml, Sigma)
agitation at 100 rpm for 16 hours. The fourth method was and RNase (10 mg/ml, Sigma) treatments of the supernatant were
cultivation under anaerobic conditions at 37uC for 16 hours with performed by incubating at room temperature for 2 hours [31].
agitation using AnaeroPack anaero (Mitsubishi Gas Chemical Co., One hundred ml of treated supernatant was used for the
Inc., Chiyoda, TKY). To determine the effect of various growth cytotoxicity assays [32]. As a control, heated TSB broth and
media on cytotoxin production, a single colony of K.oxytoca was protease-, trypsin-, DNase-, and RNAse-treated TSB broth were
inoculated into 8 ml of various types of growth media (see Media used. All assays were performed in 3 independent experiments.
Preparation) and incubated for 16 hours at 37uC under
microaerobic condition with gentle agitation. The incubation
Media preparation
duration of 16 hours was chosen based on the report that
Three different types of media: soy extract only, casein extract
cytotoxin production reaches maximum levels at the end of the
exponential growth phase, approximately 16 hours after inocula- only, and TSB, were prepared. Media containing soy extract only
tion into TSB liquid media [27]. At this stage, the liquid cultures consists of peptic digest of soybean meal (Bacto Soytone, BD;
had OD measurement range of 1.4–1.6. 20 g/L of water), dextrose (2.5 g/L), and NaCl (5 g/L). Media660 nm
containing only casein extract consists of pancreatic digest of
Casein (Bacto Tryptone; 20 g/L of water), dextrose (2.5 g/L) and
Cytotoxicin Assays
NaCl (5 g/L). TSB contains pancreatic digest of casein (Bacto
HEp-2 (ATCC-CCL-23) and/or HeLa S3 (ATCC-CCL-2.2)
Tryptone 17 g/L), Bacto Soytone (3 g/L), dextrose (2.5 g/L), and
cell lines were used to investigate cytotoxic activity in K.oxytoca as
NaCl (5 g/L).
previously described [22,23]. Initially, both HEp-2 and HeLa cells
were used to determine cytotoxity of all K. oxytoca strains. Only
HEp-2 cells were used in all other subsequent experiments. Cell Molecular weight determination
lines were maintained as recommended by ATCC with Hyclone Supernatant from the mouse clinical K.oxytoca isolate, 09-7231-
MEM/EBSS + 2.0 mM L-glutamine, + Earle’s balanced salts 1, grown under microaerobic conditions as described above, was
(HyClone Laboratories Inc., Logan, Utah) and 10% fetal bovine subjected to filtration using Centricon Ultracel YM 3 kD and
serum (Atlas Biologicals, Fort Collins, CO). Approximately 30 kD membranes (Millipore Corporation, Billerica, MA). Frac-
1.06104 cells suspended in 500 ml of cell culture media were tions obtained were then assessed in the in vitro cytotoxicity assay
seeded into 24-well cell culture plates and incubated for 1–2 hours to determine biological activity.
in 37uC with 5% CO2. To investigate cytotoxin production among
different K. oxytoca strains, 100 mL of supernatant obtained from Global gene expression analysis by microarray
liquid culture (see Bacterial Cultures) were added to the wells Human GE 4644K V2 Microarray (Ilumina) was used to
containing HEp-2 or HeLa cells. To obtain the supernatant, the investigate global gene expression changes associated with the K.
PLOS ONE | www.plosone.org 3 July 2014 | Volume 9 | Issue 7 | e100542
Klebsiella oxytoca in Laboratory Animals
oxytoca toxin on HEp-2 cells. 16106 HEp-2 cells suspended in concentrated under reduced pressure, and the crude product was
1 mL culture medium were added in each well of the 6-well tissue semi-purified by automated reverse phase flash chromatography
culture plate. Two hundred microliters of supernatant from the (Biotage) using a water/methanol gradient. The fractions eluting
cytotoxin positive K. oxytoca, 09-7231, which was subjected to 3K at 65% methanol were collected, concentrated under reduced
protein separation membrane, was added to each well. As a pressure and re-dissolved in 5% acetonitrile in water. Aliquots
control, HEp-2 cells were treated with TSB media subjected to 3K were further purified by preparative HPLC with an Agilent
protein separation membrane. Six hour post treatment, the cells Technologies model 1100 HPLC system equipped with a
were collected and RNA extracted using a combination of Trizol photodiode array UV detector (Wilmington, DE). UV absorbance
and RNA extraction kit (Qiagen). Samples from 4 independent was monitored at 254 nm. A semi-preparative Phenomenex Luna
toxin treated and control tissue culture wells were used for C18 (25 cm69.4 mm, 10 mm) column was eluted with a linear
microarray analysis. RNA samples were processed by MIT gradient of 0.1% formic acid in water (A) and 0.1% formic acid in
Biomicro Center (http://openwetware.org/wiki/BioMicroCenter). acetonitrile (B) at a flow rate of 2.5 mL/min. Solvent composition
The samples were labeled according to Agilent Two-Color was initially at 10% for 5 min, changed to 50% over 10 min,
Microarray-based Gene Expression Analysis Protocol (Low Input changed from 50% to 70% B over 15 min, and then further to
Quick Amp Labeling Kit) (http://www.chem.agilent.com/library/ 95% B over 2 min, held for 8 min, followed by returning to 10% B
usermanuals/public/G4140-90050_GeneExpression_TwoColor_6. over 2 min for a total run time of 42 min. The column was
6.pdf). RNA was hybridized to Human GE 4644 v2 microarray equilibrated for 10 minutes between injections. The fractions
(Agilent Technologies, Santa Clara, CA). Arrays were washed and eluting around 21.4 minutes (Figure S5) were collected and
scanned using the G2565 Microarray Scanner (Agilent Technolo- combined for toxicity evaluation and NMR analysis (Figure S4).
gies). For visualization, mRNA expression analysis and other
bioinformatics analyses of the microarray data, we utilized Agilent’s
Genome sequencing and In Silico Analysis
GeneSpring GX software suite. We performed differential expres-
To further explore the pathogenic mechanisms of K. oxytoca,
sion analysis using the t-test against the null hypothesis of no change
the genome of the mouse K. oxytoca strain, 09-7231-1, was fully
on each of , 34000 probes. Multiple testing correction for the
sequenced in collaboration with the Broad Institute of Harvard
differential expression analysis was done using the Benjamini-
Hochberg method. We conducted Gene Ontology functional and MIT (Klebsiella group Sequencing Project (http://www.
annotation enrichment analysis using DAVID as well as pathway broadinstitute.org/)). The full genomic sequence was deposited at
enrichment analysis in GeneSpring GX for the genes that showed the National Center for Biotechnology Information (NCBI
significant differential expression (p ,0.05). In these enrich- accession: PRJNA52135). Using a comprehensive set of referenceadjusted
ment analyses, p ,0.05 (using the Benjamini-Hochberg ‘‘heat-labile toxins’’ obtained from NCBI (n = 920), we conductedadjusted
procedure) was defined as significant. homology searches and used the BLOSUM80 scoring matrix [33],
which was created based on local alignments of highly homologous
Tilivalline detection by LC-MS and LC-MS/MS ($ 80%) proteins. We assumed that domains contributing to the
virulence and heat liability should be highly conserved between
HPLC grade chloroform and acetonitrile, formic acid for LC-
bacterial strains since these key functions are essential to their
MS, and LC-MS/MS were purchased from Sigma Aldrich. The
fitness. We varied the E-value threshold to the maximum value of
supernatant from culture broth was extracted with chloroform,
dried, and resuspended in 50 mL 2% acetonitrile containing 0.1% 10, as it is possible to have a high percentage of homologues in
formic acid. Samples were analyzed with an Agilent 1290 ultra- BLAST results with E-values ranging from 0 to 10. Indeed, a study
high pressure liquid chromatography system (Waldbronn, Ger- by Boekhorst. et al., revealed that, when querying annotated
many) interfaced with a 6530 quadrupole time-of-flight (QTOF) protein families in the PFAM database [34,35], 65% of BLAST
mass spectrometer with a Jetstream electrospray ionization source hits with E-values greater than 1e-03 and less than 10 were known
and MassHunter workstation (version B.06). The column was an homologues of the query protein according to PFAM. Addition-
Agilent C18 (2.1650 mm, 1.8 mm). Mass spectra were acquired in ally, the same study found that 43% of BLAST hits with E-values
the positive ion mode from m/z 60 to m/z 1000 at 4 scans per greater than 1 and less than 10 were known homologues [35].
second. The ion spray voltage was 3,800 V, and the heated Using the thresholding approach to prioritize hits, we aimed to
capillary temperature 350uC. Two reference masses (m/z 121.0509 include all genes that could be potential K. oxytoca cytotoxin
(C H proteins. We performed in-depth analyses of the subset of toxins5 4N4) and m/z 922.0098 (C18H18O6N3P3F24) were infused
during the runs. The column thermostat and autosampler that met an E-value cutoff of 1. In this last set of genes, we
temperatures were 50uC and 6uC, respectively. The solvents were included the toxin, PaxA, because one of its transporters, PaxB,
water with 0.1% formic acid and acetonitrile containing 0.1% had produced statistically significant results in our first round of
formic acid. The gradient was 2% to 90% B over 5 minutes, at BLAST analyses. We calculated the molecular weights of the
0.5 mL/min. subset of toxins showing homology at the chosen E-value
MS/MS was generated on an Agilent QTOF 6530 mass threshold, using the ‘‘Compute pl/Mw’’ tool on the ExPASy
spectrometer (Santa Clara, CA) to further confirm the identity of server [36].
the metabolites. The column and gradients were the same as those
used for metabolite profiling. The AJS-ESI source was set as MS Statistical analysis
scan mode. A targeted list, which included previously determined We tested whether there was a significant relationship between
exact masses according to results obtained with extracted ion the percentages of K. oxytoca strains producing cytotoxin in one
chromatogram (EIC), was generated for fragmentation. growth condition version another growth condition using a Chi-
squared test with 2 degrees of freedom.
Isolation of tilivalline
The supernatant from the growth media (1 L) was extracted in
500 mL portions, each with 250 mL of chloroform (3X). The
combined organic phase was dried (anhydrous sodium sulfate),
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Klebsiella oxytoca in Laboratory Animals
Results As expected, the non-toxigenic strain of K.oxytoca, ATCC 13182,
produced supernatant that did not have any obvious effects on
Animal isolates of K. oxytoca HEp-2 and HeLa cells (Figure 2). Compared to HeLa cells, HEp-2
Sixty four isolates from various animals were confirmed as K. cells were more susceptible to cytotoxin produced by K. oxytoca
oxytoca (Table 1). Of the 48 mouse isolates, 14/48 (29%) were isolates investigated (data not shown); therefore, HEp-2 cells were
cultured from the respiratory tract, 19/48 (40%) from feces of used in the subsequent experiments.
asymptomatic mice, 4/48 (8%) isolates from abscesses in the lung,
palpebral conjunctiva, and a tumor, and 11/48 (23%) from Growth conditions influence cytotoxin production in K.
unknown tissues or sites. Of the 4 rat isolates, 1 was isolated from oxytoca
mammary lesions, while the rest were isolated from unknown
Previous investigations on cytotoxin production in strains of K.
tissue. Three of 8 isolates of the non-human primates were oxytoca isolated from human patients were performed under static
obtained from feces; tissue origins of the 5 remaining primate
aerobic conditions i.e., in TSB liquid media incubated in 37uC
isolates, the pig and guinea pig isolates were unknown (Table 1). incubator without agitation [4,22,23]. Because K. oxytoca can
Given that K. oxytoca isolates have varying pathogenic thrive in various environmental conditions (e.g. on the skin where
potentials which could, in part, be attributed to their antibiotic O2 tension is high relative to the gastrointestinal tract), we
resistant signatures (Table 2), we determined the antibiotic hypothesized that cytotoxin production of K. oxytoca may be
resistant characteristics of each of these isolates. Overall, 27/64 differentially regulated depending on environmental condi-
(42%) of the isolates were resistant to cephalothin, 5/64 (8%) to tions[37,38,39,40]. Therefore, in addition to the static culturing
amoxicillin clavulanate, 4/64 (6%) to trimethoprim/sulfamethox- conditions commonly used (AR), we subjected all K. oxytoca
axole, 4/64 (6%) to enrofloxacin, and 2/64 (3%) to gentamicin. isolates to three additional in vitro conditions – aerobic conditions
All 4 isolates that were resistant to trimethoprin/sulfamethoxole, with gentle agitation without CO2 (AG), microaerobic conditions
enrofloxacin, and 2 isolates that were resistant to gentamicin were with gentle agitation (MG), and anaerobic conditions (AN) (See
exclusively from non-human primates. All K. oxytoca strains were Materials and Methods) – and evaluated whether these
resistant to ampicillin, a key characteristic of this b-lactamase various conditions have different effects on cytotoxin production.
organism [27] (Table 2). Gentle agitation was implemented for aerobic and microaerobic
conditions to ensure maximum exposure of bacteria to O2 in
Cytotoxin production of K. oxytoca isolates of animals aerobic conditions as well as CO2, H2, and N2 present in the
To determine whether K. oxytoca strains isolated from animals microaerobic conditions. While 10/48 (,20%) of mouse isolates
produced cytotoxin, we performed in vitro cytotoxicity assays were found to be cytotoxin positive under AR, 20/48 (,42%) and
using both HEp-2 and HeLa S3 cells (See Materials and 18/48 (,38%) were cytotoxin positive under AG and MG,
Methods). All K. oxytoca strains were grown using the previously respectively (Table 3). This increase in the number of cytotoxin
reported culturing methods [22,23], which were also described in positive strains was statistically significant between AR and AG
Material and Methods. Of the 64 isolates of K. oxytoca (p = 0.03), whereas the difference in AR and MG showed a trend
investigated, 18 (28%) isolates were found to induce greater than toward significance (p = 0.07). Of the 20 mouse isolates that were
50% cell abnormality and death (, 50% confluency), fulfilling our cytotoxin positive under AG, only 7 of these isolates were also
criteria for classifying the bacteria as cytotoxin positive (Table 3). considered as cytotoxin positive under AR (Table 3). The
Table 1. Sources and origin of Klebsiella oxytoca used in this study.
Species N = Source Tissue/Media
G. Pig 1 Diagnostic Lab Unknown
Mouse 19 Diagnostic Lab 1 Feces
Mouse 1 Diagnostic Lab 1 Lung Abscess
Mouse 12 Diagnostic Lab 1 Nasal Culture/Flush
Mouse 1 Diagnostic Lab 1 Palpebral Abscess
Mouse 6 Diagnostic Lab 1 Unknown
Mouse 2 Commercial Vendor Unknown
Mouse 2 Diagnostic Lab 2 Abscess, nu/nu mouse
Mouse 3 Diagnostic Lab 2 Unknown
Mouse 2 Diagnostic Lab 3 Nasal Flush
Rat 1 Diagnostic Lab 1 Mammary Lesion
Rat 3 Diagnostic Lab 1 Unknown
Simian 3 Diagnostic Lab 1 Feces
Simian 5 Diagnostic Lab 1 Unknown
Swine 3 Diagnostic Lab 1 Unknown
Ref. 1 ATCC 13182 Human Pharyngeal tonsil
Total 65
doi:10.1371/journal.pone.0100542.t001
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Klebsiella oxytoca in Laboratory Animals
remaining 3 mouse isolates that were cytotoxin positive under AR
were cytotoxin negative under both AG and MG. All 3 rat isolates,
a guinea pig isolate, and a pig isolate that were all cytotoxin
positive under AR were also cytotoxin positive under both AG and
MG (Table 3). In contrast, a single K. oxytoca strain isolated from
a non-human primate was positive under AR; however, it was
negative on both AG and MG. Another K. oxytoca strain from a
non-human primate was cytotoxin positive under both AG and
MG, but was cytotoxin negative under AR (Table 3).
To investigate the effects of AN on cytotoxin production, 34
selected isolates of K. oxytoca (mice: 25; non-human primates: 4;
guinea pigs: 1; pigs: 1; rats: 3), all of which produced cytotoxin
when incubated under AG and MG, were grown under AN. Only
8/34 (23%) (3 mice; 3 rats; 1 guinea pigs; 1 pig) were positive for
cytotoxin production (data not shown). Together, these results
suggest that AG and MG have a stronger positive effect on
cytotoxin production in K. oxytoca as compared to AR and AN.
Moreover, these results suggest that cytotoxin production among
different strains of K. oxytoca is under complex environmental
regulations.
Intriguingly - although perhaps coincidentally - 13/22 (59%) K.
oxytoca isolates from feces were cytotoxin positive under at least
one of the culturing conditions; virtually the same percentage (9/
14; 64%) from the respiratory tract were cytotoxin positive. Of the
four clinical isolates of K. oxytoca (3 mouse isolates isolated from
either a lung abscess, a palpebral abscess, and a tumor abscess, and
a single isolate isolated from mammary lesions in a rat (Table 1),
only one isolate (mouse isolate, 09-7231-1, from a tumor abscess)
was positive for cytotoxin.
Soy broth induces strong toxin production in K. oxytoca
Toxin production in enteric bacteria, such as Clostridium
difficile, has been shown to be regulated by nutrients [41]. To
investigate this possibility in K. oxytoca, K. oxytoca, 09-7231-1, was
grown in various growth media with varying nutrient compositions
(See Materials and Methods). This mouse isolate was chosen
because it displays the ability to produce cytotoxin consistently and
with the highest degree of cytotoxicity in the cell lines used in the
cytotoxicity assays. In addition to trypticase soy broth (TSB),
which is a standard media used to cultivate K. oxytoca and in
which toxin production has been consistently observed, LB, a
growth media with less nutrients compared to TSB, and Heart
Brain Infusion (HBI) broth, which is richer in nutrients compared
to TSB, were used. Toxin production under the three growth
media was evaluated temporally. Toxin production was found to
be highest in TSB, moderate in HBI, and lowest/nonexistence in
LB media (Figure 3).
HBI contains nutrients mainly from animal products. In
contrast, LB contains nutrient extracted from yeast. On the other
hand, nutrients present in TSB are obtained mainly from soy
product. Based on the observation that K. oxytoca grown in TSB
produce supernatant with the highest toxicity, we hypothesize that
soy product in TSB may contain compounds that contribute to the
high toxin production in K. oxytoca. Therefore, we tested the
cytotoxicity of supernatant obtained from three different media
with well-defined source of nutrients: media contains soy extract
only, media contains animal extract only (casein extract), and
media contains both soy and casein extract (TSB). Media
containing only the soy extract induces a high degree of cell
abnormality and death leading to 1% confluency among HEp-2
cells as early as 18 hours post treatment (Figure 4). As expected,
TSB media also induced cell abnormality and death in HEp-2, but
to a lesser degree (30% confluency at 18 hours post treatment
(data not shown)) compared to media containing only the soy
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Table 2. Antimicrobial resistance of K. oxytoca.
Amoxicillin/ Trimethoprim/
Species of origin (# of samples) Ampicillin Cephalothin Clavulanate Sulfamethoxaxole Enrofloxacin Gentamicin
G. pig (1) 1 0 0 0 0 0
Mouse (48) 48 23 4 0 0 0
Rat (4) 4 1 1 0 0 0
Simian (8) 8 0 0 4 4 2
Swine (3) 3 3 0 0 0 0
Antibiotic discs: ampicillin10mg; cephalothin 30mg; amoxicillin/clavulanic acid 20/10mg; trimethoprim/sulfamethoxazole 1.25/23.75mg; enrofloxacin 5mg; and gentamicin 10mg.
doi:10.1371/journal.pone.0100542.t002
Klebsiella oxytoca in Laboratory Animals
Table 3. Cytotoxin of K. oxytoca varies with different growth conditions.
Species of origin Number of strains Supernatant Growth Conditions
Aerobic w/5% CO2 (AR) Aerobic w/agitation (AG) Microaerobic w/agitation (MG)
(No. + (%)) (No. + (%)) (No. + (%))
Mouse 48 10 (20.8) 20 (41.7) 18 (37.5)
Rat 4 3 (75) 3 (75) 3 (75)
Simian 8 3 (37.5) 3 (37.5) 3 (37.5)
Swine 3 1 (33.3) 1 (33.3) 1 (33.3)
G. pig 1 1 (100) 1 (100) 1 (100)
ATCC 13182 1 0 (0) 0 (0) 0 (0)
Total 65 18 (28) 28 (45) 26 (44)
doi:10.1371/journal.pone.0100542.t003
Figure 2. Animal isolates of K. oxytoca produce cytotoxin. HEp-2 cell culture inoculated with K. oxytoca supernatant and media control at 4, 10,
and 306magnifications. A) Media control showing normal morphology of cells stained with Diff-quick after 48 hours of incubation. B) K. oxytoca,
ATCC 13182, isolate negative for cytotoxin production showing normal cell morphology. C) K. oxytoca isolate, 09-7231-1, positive for cytotoxin
production showing abnormal cell morphology, decreased concentration of attached cells, small round cells, and multinucleated cells.
doi:10.1371/journal.pone.0100542.g002
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Klebsiella oxytoca in Laboratory Animals
Figure 3. Temporal pattern of cytotoxicity in various conventional bacterial growth media. K. oxytoca, 09-7231-1, was cultured in A) LB;
B) TSB; and C) HBI liquid media. At various time points, the supernatant was collected and used in the cytotoxicity assay using HEp-2 cells. TSB media
has the strongest cytotoxin induction capability compared to LB and HBI media. The degree of cytoxicity is indirectly proportional to the level of
monolayer confluency i.e., the higher % the confluency, the lower the cytotoxity and vice versa.
doi:10.1371/journal.pone.0100542.g003
extract. Like the negative control, media containing only the have an effect on HEp-2 cells (data not shown), lessening the
casein extract did not cause any significant alteration in monolayer possibility that nutrients and components in the media are
confluency (,100% confluency). All control media also did not responsible for inducing HEp-2 cell death. Together, these results
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Klebsiella oxytoca in Laboratory Animals
Figure 4. Soy components in TSB induced HEp-2 cell detachment and death. HEp-2 cells treated with A) media containing only casein
extract (Tryptone; control); B) supernatant of K. oxytoca, 09-7231-1, grown in media containing only casein extract; C) media containing only soy
extract (Soytone); D) supernatant of K. oxytoca, 09-7231-1, grown in the media containing only soy extract.
doi:10.1371/journal.pone.0100542.g004
suggest that the soy component in TSB promotes cytotoxin Tilivalline confers cytotoxicity in vitro
production in K.oxytoca, 09-7231-1. Our studies suggested that the toxic compound was a small
endogenous and heat labile molecule. Given previous studies from
K. oxytoca cytotoxin is a small heat labile molecule human K. oxytoca isolates indicated that tilivalline is the key toxin,
It was previously demonstrated that human strains of K. oxytoca we examined the cytotoxin-positive and cytotoxin-negative strain
produced cytotoxin with an approximate size of 200 Daltons [22]. cultures for tilivalline. Comparison of LC-MS chromatograms
We investigated the size of cytotoxin produced by the mouse from control and toxic culture media (Figure S2) revealed a
isolates, 09-7231-1, using protein size exclusion and in vitro compound in the toxic media with a protonated molecular ion
cytotoxicity assays (See Materials and Methods). Both 3 KD ([M+ H+]+) at m/z = 334.1561, corresponding to a molecular
concentrate and filtrate and 30 KD filtrate cause cell monolayer weight of 333.1483 Da - within 2 ppm of that calculated for
destruction resulting in ,10% confluency, whereas 30 KD tilivalline: 333.1477 Da. CID spectra from the [M + H+]+ ion
concentrate did not have obvious effects on the cell monolayer included fragments characteristic of tilivalline at m/z = 316.1454,
(,100% confluency) (Figure S1). In addition, protease, trypsin, 219.109, 199.1237, and 136.040 (Figure S3). The proton NMR
DNase, and RNase treatment did not alter the cytotoxic activity of spectrum (Figure S4) from a culture isolate, while weak due to the
the toxic molecule present in the supernatant of K. oxytoca (data small amount of material, was also consistent with that reported
not shown); however, heat treatment completely inactivated the for tilivalline [42]. Consistent with previous reports, tilivalline
cytotoxic activity of the toxic molecule (Figure S1). There results (1 mg/ml) induced cell abnormalities and death in HEp-2 cells
suggest that the cytotoxin present in the supernatant of K. oxytoca, (Figure 5).
09-7231-2, is likely a small, heat labile compound. Furthermore,
the compound is unlikely to be RNA, DNA, or protein. Toxin-induced perturbation using mRNA profiling
Additionally, supernatant placed in room temperature for as long To investigate the molecular mechanism of toxin-induced cell
as 8 weeks exhibited strong cytotoxicity on HEp-2 cells indicating death, we performed differential expression analyses on more than
that the toxin is stable at room temperature (data not shown). 34000 probes, each representing a gene, using the Human 4644
GE v2 Agilent array. Plotting the log2 (fold change) and –log10
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Klebsiella oxytoca in Laboratory Animals
Figure 5. Tilivalline induced HEp-2 cell detachment and death. A) HEp-2 cells incubated with the control DMSO, which was used to dissolve
tilivalline. The majority of cells adhered to the bottom of the cell culture well after 48 hours of culture; B) HEp-2 cells treated with purified tilivalline
(1 mg/ml) were detached from the cell culture well after 48 hours of incubation.
doi:10.1371/journal.pone.0100542.g005
(corrected p-value) from the differential expression analysis, we synthesis, cell division and growth, stress responses, and motility.
found a large number of toxin-perturbed gene expression traits The genome also contains a gene encoding colicin, which
(see volcano plot in Figure S6, where each red point indicates a promotes bacterial fitness by killing competing bacteria through
differentially expressed gene). Table S1 lists the most significantly membrane depolarization [43].
perturbed genes (padjusted,0.05) including the direction of effect.
Notably, we found that the genes whose expression levels were Chromosomally encoded putative toxins
significantly altered by the presence of the toxin were enriched in To investigate whether other known bacterial toxin(s) besides
the following pathways: androgen receptor, hedgehog, ID tilivalline may be produced by K.oxytoca, we performed in silico
signaling, NOTCH, TGFBR, and TNF alpha/NF-kB. Some of analyses using the blastp algorithm[44] against the whole genome
the most significant Gene Ontology functional annotations (Table assembly of K. oxytoca, 09-7231-1, that we generated. Compre-
S2 for comprehensive list) include regulation of RNA metabolic hensive analysis of the genome sequence revealed genes with
process (p 235adjusted = 1.03610 ), regulation of nitrogen compound significant homology to toxin components of Vibrio cholera,
metabolic process (padjusted = 1.46610
230), negative regulation of Citrobacter freundii, and Escherichia coli (http://home.uchicago.
apoptosis (padjusted = 1.2610
23), response to DNA damage stimulus edu/,egamazon/koxytoca/index.html). Our first round of
(p 23adjusted = 1.75610 ), and cell cycle (padjusted = 0.003). BLAST analyses showed PaxB to have high homology (E-
value = 0) to hypothetical proteins in the K. oxytoca strain. PaxB
Potential virulence genes of K. oxytoca plays a role in secreting the RTX toxin, PaxA, in Pasteurella
Although the cytotoxic properties of tilivalline may in part aerogenes [45]. Although the roles of these largely uncharacterized
explain the pathogenesis of the animal isolates K. oxytoca. other K. oxytoca genes have not been fully elucidated, the high homology
pathogenic mechanisms present in the bacteria may also between the proteins suggests some type of transportation role that
contribute to its success as a pathogen. To further explore this warrants future functional validation.
possibility, the genome of a cytotoxin positive mouse isolate of K. Additionally, genes encoding proteins with high similarity to
oxytoca, 09-7231-1, was fully sequenced, and its genome was known heat-labile toxins in other enteric pathogens were identified
analyzed to determine if virulence genes identified in other (Figure 6). Two of these toxins - the cholera toxin (CT) in V.
bacteria were present. Based on current annotation, the entire cholerae, and the heat-labile toxin (LT) in enterotoxigenic E. coli
genome of K. oxytoca, 09-7231-1, is 6.17Mb in size and has the G+ strains (ETEC) - are particularly interesting. They both typically
C content of 54.78%. Nearly 88% (5,673 genes) of the genome is consist of 2 subunits—a catalytic A subunit and an immunogenic B
comprised of coding regions. Additionally, the genome contains 74 subunit [46]. BLAST analysis indicated that the K. oxytoca strain
tRNAs and 22 rRNAs. Among genes annotated as having known contained a gene with homology to a partial sequence of a known
function, genes that encode type I, type IV, and type VI secretion cholera toxin (CT) A subunit, containing the conserved domain
systems were detected. Other genes present in this strain include pfam01375 (Figure 6).
those involved in nitrate and allantoin metabolism, iron uptake,
BLAST analysis also revealed genes with homology (E-value,
resistance to various toxic and antimicrobial compounds, e.g. beta-
0.25) to CFXB from C. freundii 48. CFXB encodes a 12-residue
lactamase, macrolide, and citrate fermentation. A gene encoding
polypeptide with 73.8% sequence identity with the beta subunits of
ecotin, which is involved in host immune modulation, was also
LT and 72.8% sequence identity with the beta subunit of CT [46].
found. Evidence of transposon and phage genetic elements was
CFXB, with a molecular weight of 14.2 kDa, contains the
also detected. Genes encoding type 1 and type 3 pilli shown to be
conserved domain pfam01376 for the heat-labile enterotoxin beta
involved in biofilm formation as well as a large set of housekeeping
chain.
and structural genes were present (Table S3). These housekeeping
and structural genes include those that are involved in the
metabolism of various amino acids, fatty acids, sugars, as well as
transportation of ion, organic and inorganic compounds, DNA
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Klebsiella oxytoca in Laboratory Animals
Figure 6. The reference proteins encoded by CTA, CFX B, and PAX B are displayed as colored bars. The arrow beneath the bar shows the
region in the reference protein to which the corresponding K. Oxytoca protein aligns from the BLAST analysis.
doi:10.1371/journal.pone.0100542.g006
Discussion strains of K. oxytoca [4,22,23]. This indicates that K. oxytoca of
animals, similar to those isolated from humans, produce cytotoxin
This study investigated the possibility that animal isolates of K. that, in part, may be responsible for promoting K. oxytoca
oxytoca produce a specific cytotoxin and explored the mechanisms pathogenesis.
governing its pathogenicity with in vitro assays as well as genome Our results indicate K. oxytoca cytotoxin production is under
sequencing and analysis. To accomplish this, we characterized 64 strict environmental regulation. Specifically, microaerobic and
isolates of K. oxytoca from various animal species. Of these K. aerobic conditions i.e, AG and MG, were more effective in
oxytoca strains, 18 (28%) produced supernatant that caused HEp-2 promoting cytotoxin production among K. oxytoca strains
and HeLa cell detachment and death when grown under in vitro investigated compared to anaerobic (AN) and static aerobic
conditions previously used to identify cytotoxin-positive human conditions (AR). Although AR provides anaerobic condition
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Klebsiella oxytoca in Laboratory Animals
initially, it actually resembles an anaerobic condition after genome of K. oxytoca 09-7231. Resistance to amoxicillin/
16 hours of culture when oxygen is depleted by the growing clavuranate was also observed in select strains of K. oxytoca.
population of bacteria. The resulting anaerobic condition of AR Interestingly, K. oxytoca isolates originating from non-human
may explain the lower effect on cytotoxin production similarly primates showed resistance not only to ampicillin, cephalothin and
observed in AN as compared to AG and MG. The observation amoxicillin/clavuranate, but also to trimethoprim/sulfamethoxa-
that AG and MG have greater promoting effects on cytotoxin zole, enrofloxacin, and gentamicin. The high percentage of
production in K. oxytoca compared to AN may, in part, explain antibiotic-resistant strains of K. oxytoca isolated from nonhuman
why K. oxytoca has been reported to cause lesions in the mammary primates may indicate the liberal use of antibiotics in primates,
gland, eye, ear, respiratory and urogenital tract, where O2 tensions particularly when the animals are in quarantine and placed on
are relatively high, and diseases associated with gastrointestinal prophylactic antibiotics prior to release for research purposes [30].
tract, which offers K. oxytoca a microaerobic environment for Genomic analysis of a cytotoxin-positive K. oxytoca isolated
growth. The aerobic and microaerobic conditions these tissues from a clinically affected mouse revealed homologs of multiple
provide may favor high level of cytotoxin production and thus genes that promote pathogenicity in other pathogens. These
greater cell death. These results are not surprising as environ- include type I, type IV, and type VI secretion systems. The type IV
mental regulation of virulence genes and toxin production has also secretion system promotes pathogenicity in H. pylori through
been observed in other gastrointestinal pathogens, such as Shigella injection of cagA [50]. More recently, the type VI secretion system
spp., E. coli, Yersinia enterocolitica and Mycobacterium avium has been demonstrated to promote bacterial competition, host cell
[37,38,39]. Surprisingly, cytotoxicity of K. oxytoca was also found adhesion, and invasion in Escherichia coli, Campylobacter jejuni,
to be influenced by nutrients, and soy extract appears to have Salmonella spp [51,52]. In clinical isolates of K. pneumoniae,
promoting effects on the production of toxin. It is increasingly cultured from a liver abscess in a human, genes involved in
cited that soy products have a negative effect on gastrointestinal allantoin metabolism were highly and uniquely upregulated, while
health in animals and humans. For example, there is an increasing the non-clinical strain of K. pneumoniae lacked genes involved in
incidence of food protein-induced enterocolitis and other inflam- this metabolic pathway[53]. The mouse clinical isolate of K.
matory bowel disease entities that coincide with increased oxytoca investigated in this study also contains a large number of
consumption of soy products [47,48,49]. It is therefore possible genes involved in allantoin metabolism, including alls and allr,
that soy-induced toxin production in gut microbes, including K. which were found in the pathogenic strain of K. pneumoniae. This
oxytoca, may in part play a role in the pathogenesis of suggests that, like K. pneumoniae [53], K. oxytoca may utilize
inflammatory bowel diseases in susceptible individuals. Further allantoin metabolism to initiate extraintestinal pathology, partic-
investigations are needed to investigate this possibility and to ularly in a pathological condition when high allantoin concentra-
determine how soy products promote cytotoxin production in K. tion in the tissues is present, while other nitrogen energy sources
oxytoca. are limited [53]. The K. oxytoca isolate also contains genes
Of the 4 clinical K. oxytoca isolates obtained from laboratory involved in citrate fermentation, previously found in some clinical
animals, only one isolate was cytotoxin positive. In a related study, isolates of K. pneumoniae, as well as genes encoding ecotin. The
K. oxytoca was isolated from 14 of 200 patients (7%) being presence of ecotin, a protease inhibitor present in Yersinia pestis,
screened for vancomycin-resistant enterococci (VRE), but only one E. coli and Pseudomonas aeruginosa, known to inhibit neutrophil
of the 14 isolates (7.1%) was cytotoxic [12]. In another study, K. elastase, suggests that K. oxytoca may utilize this protein to
oxytoca was isolated from 42 of 429 patients (9.8%) tested for C. manipulate neutrophil function, thereby aiding the persistence of
difficile cytotoxicity; only 10 isolates (23.8%) were cytotoxic [12]. the organism[54]. Indeed, K. oxytoca shows a remarkable ability to
It is likely that the low percentage of cytotoxin production among persist in the gastrointestinal tract even after aggressive antibiotic
the clinical isolates of K. oxytoca in both humans and animals is therapy with antibiotics that appear to be effective against other
due to the insensitivity of the current in vitro assays that do not resident bacteria [4]. In silico analysis also reveals three genes
provide appropriate environmental cues necessary to induce present in the genome of K. oxytoca that show partial homology to
cytotoxin production in this bacterium. As demonstrated by our known toxins and toxin transporters previously identified in other
results, cytotoxin production in K. oxytoca appears to be under pathogenic bacteria, including PAXA, cholera toxin, and CFXB.
strict environmental regulation. Identifying environmental signals Further investigation is needed to determine the biological
that induce cytotoxin production in K. oxytoca may provide relevance of these toxin homologs in K. oxytoca pathogenesis.
additional insights into K. oxytoca pathogenesis and possible Initial description of a low-molecular-weight (,217 Da) cyto-
therapeutic strategies. toxin, produced by cultured strains of K. oxytoca isolated from
The majority of mouse isolates of K. oxytoca in this study were humans with hemorrhagic diarrhea, was reported by Minami and
cultured from the feces or respiratory tract of sentinel rodents co-workers, although its structure was not characterized [22,23].
during routine health screening. This suggests that the respiratory Mohr and Budzikiewicz had earlier identified a low molecular-
and gastrointestinal tract may be the preferred site of colonization weight (333 Da) cytotoxic compound, produced by K. pneumoniae
by K. oxytoca in mice. However, the fact that a few isolates of K. serova oxytoca, as tilivalline [24]. Here we demonstrate that
oxytoca were isolated from other tissues, including palpebral cultured animal isolates of K. oxytoca also produce high levels of
conjunctiva, tumor, and, mammary gland, indicates that K. tilivalline. The purified compound was tested in vitro for cytotoxic
oxytoca can also colonize other organs. properties and was found to induce cell abnormality and death in
Antibiotic resistance patterns appear to vary considerably HEp-2 cells confirming its cytotoxic property.
among different strains of K. oxytoca. Strains of K. oxytoca are Previous studies to determine the molecular mechanisms of
resistant to amino-penicillins and carboxy-penicillins due to the toxin induced cell death in K. oxytoca suggest that the mechanism
production of b-lactamases. Antimicrobial susceptibility testing is primarily through inhibition of DNA synthesis [22,23]. Our
indicated that all of the K. oxytoca strains investigated were whole transcriptomic profiling reveals that tilivalline induced
resistant to ampicillin, suggesting these isolates of K.oxytoca are global perturbation of genes involved in numerous fundamental
able to synthesize B-lactamases. This is consistent with the cellular processes, including cell division, nitrogen metabolism,
presence of beta-lactamases that encode a gene present in the RNA metabolic processing, DNA damage repair processes,
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Klebsiella oxytoca in Laboratory Animals
apoptosis, and androgen signaling, as early as 6 hours after HEp-2 Figure S5 HPLC chromatogram with UV/vis detection
cells were exposed to the toxin when cell abnormalities are not yet for the isolation oft tilivalline (24.3 min).
observed. Although the primary target of tilivalline may be in the (TIF)
synthesis of DNA, our transcriptome data highlight the possibility
Figure S6 A volcano plot illustrated differentially regu-
of simultaneous effects of tilivalline on multiple cellular processes lated genes.
and pathways. Further investigations are required to determine the (TIF)
exact mechanism of tilivalline-induced cell death.
In conclusion, this study provides evidence that - similar to K. Table S1 The most significantly perturbed genes (ad-
oxytoca strains in humans - animal isolates of K. oxytoca are justed p-value ,0.05). In addition to the p-value from the
capable of producing cytotoxin in vitro. The roles that K. oxytoca differential expression analysis, log-fold change and direction of
cytotoxin play in induction of genitourinary diseases or abscesses effect are shown.
in other tissues in mice and its pathogenic potential in other (XLSX)
animals remain to be defined. Table S2 The most significant Gene Ontology function-
al annotations. The most significant functional annotations
Supporting Information implicated by the differentially expressed genes include regulation
of RNA metabolic process (p = 1.03610235adjusted ), regulation of
Figure S1 Cytotoxicity of supernatant on HEp-2 cells nitrogen compound metabolic process (p 230adjusted = 1.46610 ),
subjected to A) 30K concentrate; B) 30 K filtrate; C) 3K negative regulation of apoptosis (padjusted = 1.2610
23), response to
concentrate; D) 3K filtrate; E) heat-treated supernatant DNA damage stimulus (p 23adjusted = 1.75610 ), and cell cycle
of K. oxytoca, 09-7231-1; F) supernatant of K. oxytoca,
(padjusted = 0.003).
09-7231-1, without heat treatment. Note the low confluency (XLSX)
of B), C), D), and F) suggesting strong cytotoxic activity.
(TIF) Table S3 Potential virulence genes found in K. oxytoca,
09-7231-1".
Figure S2 LC/MS total-ion chromatograms. A) soy broth (DOCX)
extract; B) soy broth extract from 9-hour toxic culture; C) soy
broth extract from 24-hour toxic culture; D) soy broth extract Acknowledgments
from 16-hour culture of negative strain (non-toxic). Inset:
background-subtracted mass spectrum of the compound eluting We thank the following for providing strains of K. oxytoca: Richard Fister,
near 2.5 minutes. Charles River Laboratories, Research Animal Diagnostic Services, Julius
E. Thigpen and Tanya Whiteside, Department of Health and Human
(TIF)
Services, National Institute of Health, National Institute of Environmental
Figure S3 MS/MS spectrum from m/z 334.156 (colli- Health Sciences, Research Triangle Park, NC, and Ray A. Vonder Haar,
sion energy = 20 V), with suggested fragment struc- The Jackson Laboratory, Bar Harbor, ME, USA. We thank Alyssa
tures. Terestre for assisting with manuscript preparation.
(TIF)
Author Contributions
Figure S4 1H NMR spectrum. Recorded on a Varian Inova-
Conceived and designed the experiments: AD KL U. Sarkar ERG SRT
500 instrument operating at 500.13 MHz using 5 mm O.D. thin-
JSW JGF. Performed the experiments: AD KL U. Sarkar ERG ZS NST
walled precision NMR tubes (Wilmad). Chemical shifts are relative CC CB DP U. Seneviratne JGF. Analyzed the data: AD KL U. Sarkar
to pyridine-d5 using Varian 5 mm PFG-probes at 22uC. ERG ZS NST EMB CC CB U. Seneviratne JGF. Wrote the paper: AD
(TIF) KL U. Sarkar ERG JGF.
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