Cereus 3

RESEARCH ARTICLE
Bacillus cereus, a serious cause of nosocomial

infections: Epidemiologic and genetic survey
Benjamin Glasset1,2, Sabine Herbin2, Sophie A. Granier2, Laurent Cavalié3,
Emilie Lafeuille4,5, Cyprien Guérin6, Raymond Ruimy7, Florence Casagrande-Magne7,
Marion Levast8, Nathalie Chautemps8, Jean-Winoc Decousser9, Laure Belotti10,
Isabelle Pelloux11, Jerôme Robert4,5, Anne Brisabois2, Nalini Ramarao1*
1 Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, 2 Université
Paris-Est, Anses, Laboratory for Food Safety, Maisons-Alfort, France, 3 CHU Toulouse, Service de
Bactériologie-Hygiène, IRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, France,
4 Sorbonne Universités, UPMC Univ Paris 06, Inserm, U1135, Centre d’Immunologie et des Maladies
Infectieuses (CIMI-Paris), Paris, France, 5 Laboratoire de Bactériologie-Hygiène, Hôpitaux Universitaires
Pitié-Salpêtrière-Charles Foix, APHP, Paris, France, 6 MaiAGE, INRA, AgroParisTech, Université Paris-
Saclay, Jouy-en-Josas, France, 7 CHU Nice, Laboratoire de bactériologie, Nice, France, 8 Hôpital de
a1111111111 Chambéry, Laboratoire de Biologie Médicale, Chambéry, France, 9 Hôpitaux Universitaires Paris-Sud
a1111111111 Antoine Béclère, Laboratoire Hygiène, Clamart, France, 10 CHU Strasbourg, Laboratoire d’hygiène
a1111111111 hospitalière, Strasbourg, France, 11 CHU Grenoble, Laboratoire de Bactériologie, Grenoble, France
a1111111111
a1111111111 * [email protected]
Abstract
OPEN ACCESS Bacillus cereus is the 2nd most frequent bacterial agent responsible for food-borne out-
Citation: Glasset B, Herbin S, Granier SA, Cavalié L, breaks in France and the 3rd in Europe. In addition, local and systemic infections have been
Lafeuille E, Guérin C, et al. (2018) Bacillus cereus, a reported, mainly describing individual cases or single hospital setting. The real incidence of
serious cause of nosocomial infections:
Epidemiologic and genetic survey. PLoS ONE 13
such infection is unknown and information on genetic and phenotypic characteristics of the
(5): e0194346. https://doi.org/10.1371/journal. incriminated strains is generally scarce. We performed an extensive study of B. cereus
pone.0194346 strains isolated from patients and hospital environments from nine hospitals during a 5-year
Editor: Theresa M. Koehler, University of Texas study, giving an overview of the consequences, sources and pathogenic patterns of B.
Medical School at Houston, UNITED STATES cereus clinical infections. We demonstrated the occurrence of several hospital-cross-con-
Received: September 1, 2017 taminations. Identical B. cereus strains were recovered from different patients and hospital
Accepted: March 1, 2018
environments for up to 2 years. We also clearly revealed the occurrence of inter hospital
contaminations by the same strain. These cases represent the first documented events of
Published: May 23, 2018
nosocomial epidemy by B. cereus responsible for intra and inter hospitals contaminations.
Copyright: © 2018 Glasset et al. This is an open Indeed, contamination of different patients with the same strain of B. cereus was so far
access article distributed under the terms of the
Creative Commons Attribution License, which
never shown. In addition, we propose a scheme for the characterization of B. cereus based
permits unrestricted use, distribution, and on biochemical properties and genetic identification and highlight that main genetic signa-
reproduction in any medium, provided the original tures may carry a high pathogenic potential. Moreover, the characterization of antibiotic
author and source are credited.
resistance shows an acquired resistance phenotype for rifampicin. This may provide indica-
Data Availability Statement: All relevant data are tion to adjust the antibiotic treatment and care of patients.
within the paper and its Supporting Information
files.
Funding: The authors received no specific funding

for this work.
Competing interests: The authors have declared

that no competing interests exist.
PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 1 / 19

Bacillus cereus induces severe nosocomial infections
Introduction
Bacillus cereus is a spore forming and ubiquitous bacterium present in soil, foods, insect larvae,
almost all surfaces and human skin [1, 2]. Besides food poisoning [3, 4], B. cereus induces local
and systemic infections [5–13]. The main described conditions are septicemia, endophthalmi-
tis, pneumonia, endocarditis, meningititis and encephalitis, especially in immunosuppressed
individuals such as neonates, resulting in the patient death in about 10% of cases [9, 14–19]. In
addition, several cases of fulminant infections similar to anthrax, and affecting healthy persons,
have also been reported [20–22]. Predisposing factors include intravenous drug use, surgical
or traumatic wounds, intravascular catheters and prematurity due to an immature immune
response and to the presence of indwelling devices in the intensive care environment of neo-
nates [16–18, 23]. Environmental reservoirs include air filtration/ventilation equipment, linen,
medical devices and hands of the staff [9, 24]. Case reports describe mainly individual cases or
come from single hospital centers and no large survey has been done on B. cereus clinical infec-
tions. In addition, information on the genetic and phenotypic characteristics of the incrimi-
nated strains is generally scarce. An appropriate empirical antibiotic therapy should be started
immediately after suspicion of B. cereus infection. However, as B. cereus is mainly considered
as an environmental contaminant, delays in treatments may compromise the clinical outcome.
We performed a thorough description of clinical cases, together with an accurate pheno-
typic and genetic characterization of the strains. This should be of major interest to improve
treatments of patients with non-gastrointestinal B. cereus infections.
Material and methods

Data collection
Epidemiological and clinical data on B. cereus samples isolated from patients were retrospec-
tively collected from French voluntary hospitals between 2008 and 2012. Nine hospitals local-
ized in different regions of France participated to this non-exhaustive study. The authors did
not have access to any patient identifying information as part of this work. Each hospital filled
a questionnaire and reported every cases of patient for which B. cereus was isolated in at least
one biological sample. B. cereus strains were locally identified by plating on specific agar media
(Mossel Medium) and confirmed by using 16S rDNA sequencing. These data allowed to clas-
sify the strains as belonging to the B. cereus group and excluded B. anthracis strains. Data
included basic demographic data, hospital wards, type of clinical sample, date of sampling,
clinical data, antibiotic therapy and outcome.
In addition, B. cereus strains obtained from surface samples around clinical cases were also
included in the microbiological analysis.
Biochemical analysis
All strains were tested for their capacity to hydrolyze starch on Plate Count Agar, their hemo-
lytic activity on blood sheep agar plates and their lecithinase activity on Mossel medium as pre-
viously described [2, 25, 26].
Molecular analysis
M13 sequence-based polymerase chain reaction (M13-PCR) is derived from an RAPD tech-
nique that allows differentiating between various strain patterns. M13 typing was performed
as described [3]. The DNA profiles were analyzed with BioNumerics 7.1 software (Applied
Maths). The software compared the DNA profiles and clustered the strains according to their
similarity.

The toxin gene profiles were identified by assessing the presence of the cytK-1, cytK-2,
HBLA, HBLC, HBLD, NHEA, NHEB, NHEC, hlyII and ces genes by PCR using specific primers
[3] (S1 Fig). The strains were then clustered into genetic signatures (GS) according to their
different combinations of presence/absence patterns. We noted a potential discrepancy for 2
strains isolated from the same patient (18), one of which was positive for the ces gene and the
other one negative. Similarly, two other strains (from patients 16 and 17), which showed other-
wise identical profiles, were negative for the ces gene. This might reflect a non recognition of
the primers for these specific strains.
The strains were affiliated to the seven known phylogenetic groups according to the partial
sequencing of the panC gene [27]. The complete sequence of the panC gene was used to com-
pare the strains in the same cluster.
Genomic divergence estimation

Genomes from eight bacterial isolates were sequenced using Illumina NextSeq500 paired-end
100 bp sequencing technology with 12.5 million reads per sample. For the purpose of SNP call-
ing, B. cereus ATCC 10987 (NC_003909.8) was selected as a reference after computing Average
Nucleotide Identity (ANI) (http://enve-omics.ce.gatech.edu/ani/) between preliminary de
novo assemblies for the eight samples and several complete genomes available in the public
databases (~95% ANI between NC_003909.8 and our samples, which is comparable to the
divergence between our samples). Sequencing adaptors were removed from the reads using
Cutadapt (version 1.8.3; with options -n 5 -O 3 -m 0 options). Low quality sequence data
were trimmed using Sickle (https://github.com/najoshi/sickle) (-n -q 20 -l 20). Read mapping
on the reference was performed using Bwa mem (http://bio-bwa.sourceforge.net/bwa.shtml)
(v0.7.12-r1039; default options). Mapping depth excluding multiple mapped reads was
extracted using Samtools (v1.2) depth (-Q 1) and the core genome was defined as positions
with mapping depth > = 10 in the eight samples (which represented 3463800 bp). SNP calling
was performed using Samtools mpileup and Bcftools call (-vmO v) and variants with a quality
above 250 were selected. Pairwise divergence between samples was calculated as the propor-
tion of variable positions along the core genome. A tree depicting the relationships between
our eight samples was obtained by hierarchical clustering based on the matrix of pairwise
divergences using R (http://www.R-project.org/) function hclust.
All raw reads generated were submitted to the European Nucleotide Archive (http://www.
ebi.ac.uk/ena/) under the study accession number PRJEB18787.
Toxin production
The production of the enterotoxins NHE and HBL was tested with the immunological tests
BCET-RPLA Toxin Detection (Oxoïd) and Tecra (BDE VIA, 3M-Tecra) kits, respectively [28].
The production of NHE enterotoxin was semi-quantitatively assessed. According to the values
obtained and following the manufacturer recommendation, the NHE production was scored
as high (4–5), medium/weak (2–3) or not detectable (0–1). The production of HBL enterotoxin
was quantitatively assessed and scored according to the dilution showing an activity as highly
producer (1/64-1/32-1/16) or medium/weak producer (1/8-1/2).
Antibiotic susceptibility
The Minimum Inhibitory Concentrations (MICs) of selected antimicrobial agents were mea-
sured by using concentration gradient strips (Etest1, BioMerieux). Briefly, inoculum was
adjusted to 0.5 McFarland before being swabbed on a Mueller-Hinton agar plate (Bio-Rad).
Incubation was performed at 35˚C for 16–18 hours. The following agents were tested:

ampicillin$, cefotaxime, imipenem$, vancomycin$, gentamicin$, rifampicin$, tetracycline$, cip-

rofloxacin$, chloramphenicol$, azithromycin, sulfamethoxazole/trimethoprim$ and clindamy-
cin$. Due to scarce availability of interpretative criteria in the literature, clinical breakpoints
were used when available ($) [29].
Molecular typing and statistical analysis

For each strain, all results were entered into a central database using BioNumerics (BN) soft-
ware. A phylogenetic tree and a dendrogram from pairwise similarity matrices were built
based on panC sequence alignments and M13-PCR molecular typing, respectively using
UPGMA (Unweighted Pair Group Method with Arithmetic Mean). The percentage of identity
between strains corresponding to the mean of the three experiments was used to construct the
dendogram of Fig 1.
Data were analyzed by the FactoMineR package of the R 2.13.0 software (http://www.
agrocampus-ouest.fr/math/). Principal Component Analysis (PCA) transforms a set of puta-
tive correlated variables into new variables, which are mutually orthogonal (uncorrelated)
linear combinations of the original variables. These new variables are called principal compo-
nents (PC). Each PC is defined by the coefficients in the linear combination of the original var-
iables. For PCA, quantitative values were used for toxin production and age of patients. Each
patient’s isolate was represented by a point whose coordinates corresponded to the scores
contributing to the PC. The variable corresponding to the different Genetic Signatures was
considered as qualitative variable. A hierarchical clustering was performed on the PC (HCPC
function of FactoMineR package), in order to identify subsets of objects that corresponded to
clusters having similar characteristics within the whole collection.
Simpson index of discrimination (D) was calculated according to the equation provided in
[30], where N represents the total number of strains (N = 56) and nj represents the number of
strains belonging to each typing sub-group.
Results
Epidemiology
Nine hospitals reported 39 patients with B. cereus strains isolated in at least one clinical sample
during the five-year study period. For the microbiological analysis, a single B. cereus strain was
included per patient, except if several strains were isolated in different clinical sites (patient 1)
or over a prolonged time period (patients 18 and 19). It resulted that 45 B. cereus strains were
further analyzed, in addition to 11 strains isolated from hospital surface samples (Table 1, sev-
eral samples were isolated from patients 1, 18 and 19).
A majority of strains (41%) were isolated in newborns, among which 3/4 were premature
infants with low birth weight. Patients over 60 year old represented the second most frequent
group of patients (26%), followed by middle aged patients (23%). Wards of hospitalization and
symptoms recorded were diverse (Tables 1 and 2).
B. cereus infections led to local and systemic infections (Table 2). Local infections repre-
sented 8% of the cases. A total of 28 (72%) patients had a positive blood culture for B. cereus.
Among them, 15 had another body site displaying B. cereus including the lungs (n = 7) or the
central nervous system (n = 5). The gastrointestinal tract represented 18% of the clinical sites.
15% patients had at least three clinical sites positive with B. cereus. Death occurred in eight
(21%) patients, including four premature babies.
It is noteworthy that, for 62% (n = 24) of patients, B. cereus was considered as the potential
cause of infections and usually taken into account by the physicians for the antibiotic therapy.
In the remaining cases, B. cereus was initially wrongly considered as a contaminant.

Fig 1. Dendogram and strain data. Left panel, dendrogram obtained by cluster analysis of M13-PCR fingerprint
patterns of the 56 strains. The UPGMA was used to build a dendrogram from a pair wise similarity matrix. Seven
clusters were obtained with strains sharing 100% of homology. Right panel, data include for each strain the
corresponding patient and hospital, genetic signature, phylogenetic panC group, NHE and HBL indice, and the
antibiotic susceptibility profile. nd: not detected.
https://doi.org/10.1371/journal.pone.0194346.g001

Table 1. Epidemiological and clinical data of patients and samplings.

Hospitals, n 9
Strains, n 56
Patients, n 39
Environment sample, n 11
Male patients, n (%) 23 (59%)
Immunocompromised, n (%) 23 (59%)
Death, n (%) 8 (21%)
Age, n (%)
Premature newborn 12 (31%)
Newborn 4 (10%)
1–25 3 (8%)
26–59 9 (23%)
60 + 10 (26%)
Unknown 1 (2%)
Ward, n (%)
Neonatology 13 (33%)
Intensive care unit 6 (15%)
Medical 5 (13%)
Hematology and Oncology 5 (13%)
Surgery 4 (10%)
Emergency room 2 (5%)
Bacteriology laboratory 2 (5%)
Mortuary 1 (3%)
Unknown 1 (3%)
Environmental sampling, n (%)
Surface of neonatology ward 6 (55%)
Incubator heater 3 (27%)
Milk on gastric feeding tube 1 (9%)
Catheter for sonogram 1 (9%)
The collection contains 56 strains from nine hospitals, 45 strains isolated from 39 patients and 11 strains collected on
surface samples.
https://doi.org/10.1371/journal.pone.0194346.t001
Biochemical identification
Among the isolates characterized as B. cereus group strains, 48% presented the ability to hydro-
lyze starch, 93% had lecithinase activity and 71% were hemolytic. These data are consistent
with previous finding showing that not all B. cereus strains are hemolytic [3]. The production
of two main toxins, NHE and HBL, was assessed. 25% of the strains were high producers of
NHE and high producers of HBL, 54% were high producers of NHE and low or no producers
of HBL, 7% were low producers of NHE and high producers of HBL, and 14% were low or no
producers of NHE and HBL (Fig 1).
Molecular characterization
The presence of ten genes suspected or shown to play a role during B. cereus pathogenesis was
investigated. The combination of these genes allowed clustering the strains into ten genetic sig-
natures (GS) (S2 Fig). The cytK1 gene was not found in the strain collection.

Table 2. Characteristics of patients or hospital environment displaying B. cereus positive samples.

Sampling Hospital Date of Hospital ward Age of Type of sampling Symptoms Antibiotic Outcome
sampling patient treatment
Patient 1 Hospital 28/07/ neonatology Premature blood culture meningitis, infection in the liver, death
A 2009 newborn both lungs
Patient 1 Hospital 28/07/ neonatology Premature cerebrospinal meningitis, infection in the liver, death
A 2009 newborn fluid both lungs
Patient 2 Hospital 16/06/ neonatology Premature blood culture brain abscess VAN, CTX recovery
A 2009 newborn
Patient 3 Hospital 05/07/ neonatology Premature blood culture bacteremia VAN recovery
A 2009 newborn
Patient 4 Hospital 30/06/ neonatology newborn neonatal gastric bacteremia VAN recovery
A 2009 liquid
Patient 5 Hospital 21/07/ neonatology newborn Umbilical local colonization CTX, AMX, AMK recovery
A 2009
Hospital Hospital 23/07/ neonatology Surface of
environment 1 A 2009 neonatology
ward (Window
sill)
ward (Window
sill)
ward (Delivery
room)
ward (air vent)
Patient 6 Hospital 03/09/ neonatology newborn axilla later feces skin infection CRO recovery
B 2009
Patient 7 Hospital 17/09/ neonatology premature stomach tube premature birth CTX, AMK, AMX recovery
B 2009 newborn feeding (3 days)
Patient 8 Hospital 20/09/ neonatology premature gastric acid neonatal infection VAN (7days) recovery
B 2009 newborn
Patient 9 Hospital 21/09/ neonatology premature central venous bacteremia AMX, AMK (3 recovery
B 2009 newborn catheter days), then VAN
(18 days)
environment 5 B 2009 neonatology
ward
Patient 10 Hospital 02/08/ neonatology premature blood culture refractory hypoxemia, chronic CTX, VAN, AMK recovery
C 2011 newborn bronchial dysplasia, stage-ii (10 days)
intraventricular hemorrhage, sepsis
Patient 11 Hospital 08/2011 neonatology premature blood culture apnea, bradycardia, and gray Death
C newborn complexion. after that, sepsis, organ
failure and pulmonary and cerebral
abscesses [18]
Hospital Hospital neonatology milk on stomach
environment 6 D tube feeding
Patient 12 Hospital 06/2009 emergency 80 Thoracentesis pulmonary infection AMX
D
Patient 13 Hospital 12/2010 neonatology premature stomach tube abdominal distension followed by VAN, CTX, MTZ recovery
D newborn feeding severe enterocolitis and biological
abnormalities [17]
(Continued)

Table 2. (Continued)
Patient 14 Hospital 12/2010 neonatology premature stomach tube abdominal distension appeared VAN, CTX, MTZ recovery
D newborn feeding three days after birth associated
with radiologic, clinical, and
biologic signs of enterocolitis
Patient 15 Hospital 18/09/ intensive care unit 30 blood culture endocarditis associated to GEN, OXA (4 death
E 2011 of Tropical and methicillin- sensitive days)
Infectious Diseases Staphylococcus aureus (MSSA) in
an intravenous drug abuser, and
cerebral mycotic aneurysms
Patient 16 Hospital 02/11/ hematology 65 blood culture sepsis causing death in a very death
E 2009 pejorative context (leukocytes 0.3,
platelets 20)
Patient 17 Hospital 12/09/ nephrology 54 blood culture sepsis and undernourishment VAN, CRO, then recovery
E 2011 VAN, CIP (21
days)
Patient 18 Hospital 03/03/ gastroenterology 63 blood culture bacteremia and central venous AMX, then CIP recovery
E 2010 catheter-linked infection (21 days), then
GEN (3days), IPM
(18days), then
CIP, VAN (10
days)
GEN (3days), IPM
(18days), then
CIP, VAN (10
days)
GEN (3days), IPM
(18days), then
CIP, VAN (10
days)
Patient 19 Hospital 01/12/ hematology 61 blood culture sepsis (patient with an acute PIP, AMK, VAN recovery
E 2010 myeloid leukemia) (7 days), then CIP,
GEN
Patient 19 Hospital 07/12/ hematology 61 blood culture sepsis (patient with an acute PIP, AMK, VAN recovery
E 2010 myeloid leukemia) (7 days), then CIP,
GEN
Patient 20 Hospital 03/06/ surgery 34 blood culture bacteremia (drug addict patient recovery
E 2008 with axillary abscess)
Patient 21 Hospital 27/11/ neurology newborn blood culture kidneys and urinary infections CRO, GEN recovery
E 2010
Patient 22 Hospital 15/06/ neurology 43 blood culture bacteremia recovery
E 2008
Patient 23 Hospital 06/10/ oncology 66 blood culture bacteremia (patient with a colorectal recovery
E 2009 cancer)
Patient 24 Hospital 24/09/ hematology 24 blood culture sepsis and aplastic anemia caused by PIP, AMK recovery
E 2010 + skin infection drugs
Patient 25 Hospital 12/08/ gynecological 77 blood culture bacteremia (patient with breast CIP recovery
E 2009 surgery cancer)
Patient 26 Hospital 16/07/ cardiac surgery 60 blood culture sternum abscess, absent fever Sequela of
E 2010 osteitis
Patient 27 Hospital 20/06/ hematology 40 blood culture bacteremia (immunocompromised recovery
E 2008 patient)
(Continued)

Table 2. (Continued)
Patient 28 Hospital 07/2011 orthopedic surgery 31 Prosthesis from no clinical sign of infection AMX recovery
F tibia
Patient 29 Hospital 10/2011 intensive care unit 76 blood culture community acquired pneumonia CTX, SPI then recovery
F CTX
Patient 30 Hospital 09/2012 intensive care unit 46 catheter culture heart failure and multiple infectious VAN, CLO, GEN recovery
F without an blood episodes then AMX then
positive culture PIP then IPM then
IPM, CAZ, CIP
Patient 31 Hospital 09/2012 intensive care unit 48 blood culture acute respiratory distress syndrome CRO, GEN then recovery
F CAZ, then PIP
then CAZ, VAN,
AMK
Patient 32 Hospital 06/2011 intensive care unit 86 blood culture heart failure, ventilator-associated AMK, IPM then recovery
F from catheter pneumonia, ischemic stroke IPM
Patient 33 Hospital 10/2011 emergency 24 blood culture abdominal pain, shivering, none recovery
F vomiting, fever, diarrhea
Patient 34 Hospital 10/2012 intensive care unit 56 blood culture bronchogenic carcinoma, CTX then PIP death
F from catheter pneumonia then AMK, IPM
Patient 35 Hospital 09/2012 gastroenterology 85 Liver abscess sepsis, hepatitis c and liver abscess, GEN, CTX, then recovery
F abdominal pain, diarrhea CTX, CIP, then
SXT, OFX, CTX
Patient 36 Hospital 09/2013 ? blood culture nausea, abdominal pain and ?
G vomiting
Hospital Hospital clinical laboratory babies
environment 7 H environment
Hospital Hospital clinical laboratory environment of
environment 8 H incubator heater
Hospital Hospital clinical laboratory Incubator
environment 9 H environment
Hospital Hospital clinical laboratory Catheter for
environment H sonogram
10
Hospital Hospital clinical laboratory Incubator
environment H environment
11
Patient 37 Hospital 12/2013 clinical laboratory Premature Blood culture septic shock, multiple organ failure, VAN death
H newborn from umbilical pulmonary and cerebral abscesses
venous catheter
Patient 37 Hospital 12/2013 clinical laboratory Premature blood culture septic shock, multiple organ failure, VAN death
H newborn from peripheral pulmonary and cerebral abscesses
veins
Patient 38 Hospital 12/2013 clinical laboratory Premature Bronchial septic shock and pneumonia VAN death
H newborn aspiration (lung) pulmonary necrotic abscesses,
recurrent pneumothorax
Patient 39 Hospital 2014 ? Biopsy (kidney) vomiting and diarrhea death
I
Patient 39 Hospital 2014 ? Biopsy (spleen) vomiting and diarrhea death
I
Data included hospital wards, date of sampling, patient age, type of sample, infection sites, clinical data, antibiotic therapy and outcome.
CTX: cefotaxime, VAN: vancomycin; AMK: amikacin; AMX: amoxicillin; MTZ: metronidazole; OXA: oxacillin; CRO: ceftriaxone; CIP: ciprofloxacin; IPM: imipenem;
PIP: piperacillin; CAZ: ceftazidime; CLO: cloxacillin; SXT: cotrimoxazole; OFX: ofloxacin; GEN: gentamicin; SPI: spiramycin
https://doi.org/10.1371/journal.pone.0194346.t002

The most frequent GS were GS1 (nhe only, 34%), GS2 (nhe, hbl, cytk-2, 23%), GS3 (nhe, ces,
16%) and GS4 (nhe, cytk-2, 14%) (Fig 1).
Our clinical strains belonged to only three phylogenetic groups by panC sequence analysis
II, III and IV representing 23%, 47% and 30% of the strains, respectively (Fig 1).
Finally, the M13 pattern of each strain was assessed. A dendogram from pair wise similarity
matrixes was build based on M13-PCR molecular typing using UPGMA (Unweighted Pair
Group Method with Arithmetic Mean). 41 different M13 profiles were identified according
to the percentage of identity between strains (Fig 1). The discriminating Simpson’s index
revealed that M13 PCR allowed a high power of differentiation of the strains (discrimination
index 0.983).
Antimicrobial susceptibility
The Minimum Inhibitory Concentrations (MICs) of selected antimicrobial agents were mea-
sured. According to CLSI clinical breakpoints, the tests revealed five susceptibility patterns
(Figs 1 and 2 and S3 Fig). Natural resistance to beta-lactams was confirmed for ampicillin and
Fig 2. MIC results (Etest method) for the 56 B. cereus strains. Black lines: population distribution. Concentrations indicated in red are classified as clinically
resistant according to CLSI or EUCAST (no known values for Azithromycin, cefotaxime and vancomycin).

cefotaxime, while imipenem appeared active at low concentrations. All strains were catego-
rized as susceptible to vancomycin, gentamicin, tetracyclin, ciprofloxacin, azithromycin and
clindamycin. Two strains were resistant to chloramphenicol. One strain was resistant to rifam-
picin and one to cotrimoxazole (trimethoprim/sulfamethoxazole), respectively.
Principal component analyses

To analyze the potential correlations between the phenotypic and genotypic characterizations
of the strains, principal component analyses were performed for each characteristic. There
were no obvious correlations between GS and/or symptoms, hospital wards and patient age. By
contrast, several clusters of strains appeared when considering in the PCA the three compo-
nents: age of patient, NHE production and HBL production (Fig 3). The circles of correlations
indicate the strains that can be statistically grouped. Strains producing high level of HBL were
on average weak producers of NHE (strains clustering in the red circle). Strains producing high
level of NHE infected middle age or elderly patients (>34 year old) (blue circle). There was
no correlation with strains weakly producers of HBL and NHE production or age of patients
Fig 3. Correlation clusters of the quantitative variables characterizing each B. cereus strain isolated from patients. The percentages of
variation explained by the principal components (PC1 and PC2) are indicated in brackets. The factors involved in PC1 (Dim1: age of patients
and NHE indice) and PC2 (Dim2: HBL indice) are indicated in the variable factor map at the top right of the figure. The strains located inside a
colored circle belong to the same cluster, as determined by the hierarchical cluster analysis performed after PCA. Each dot corresponds to a
strain. The squares represent the representative value for the cluster.

(green circle). The most striking results was the correlation between the low age of the patient
(<6.5 year old) and no or weak production of HBL and NHE (strains in the black circle).
Molecular epidemiology
Intra-hospital contaminations. We identified eight cases of intra-hospital cross infec-
tions: the occurrence of an infection by a single strain of B. cereus of at least two different
patients within the same hospital. Strains with identical M13 pattern (100% identity by
UPGMA on dendogram), panC sequencing (over the entire gene), GS, toxin production and
antibiotic susceptibility/resistance pattern were grouped in a single molecular profile (Fig 1).
Strains with identical molecular profiles were recovered from different patients and hospitals
strongly suggesting the occurrence of eight hospital cross-infections. Fig 4 shows the M13 pro-
files (top level) and the WGS-based pairwise divergence (bottom) of the strains involved in the
Fig 4. M13-PCR fingerprint patterns of B. cereus strains showing eight possible cross contaminations between patients/patients or
patients/environment. Top panel, lane 1: 1kb DNA ladder. Lane 2: reference strain B. cereus ATCC14579. Lane 2 to 23: B. cereus strains.
Bottom: divergence tree between eight samples obtained by hierarchical clustering based on the matrix of pairwise divergences after WGS data.

8 cases of cross-infections. For M13 profiles, Fig 4 (top) shows one representative image but
the calculated % of identity between strains is shown on the dendogram of Fig 1.
At hospital (A), a B. cereus strain was isolated from the umbilical cord of a newborn (patient
5). Two B. cereus isolates with identical profiles were isolated one week later from two clinical
samples (blood culture and cerebrospinal fluid) from another neonate (patient 1). Patient 1
died at day 6 following a sepsis, and multiple-site infection with no antibiotherapy. The three
isolates of patients 1 and 5 were characterized by the M13-1 profile, GS1, panC group III,
100% identity over the panC gene sequence, a medium NHE and low HBL production and the
antibiotic profile a. All these similarities, and especially the M13 pattern, strongly suggested
that the three isolates were identical and/or may descend from a same recent ancestor. To
confirm these findings, the three genomes of the strains were entirely sequenced (WGS). The
divergence tree between the strains was obtained by hierarchical clustering based on the matrix
of pairwise divergences. The three genomes showed 100% identity (Fig 4 and S4 Fig). Indeed,
1 or 2 SNP differed between the strains and pairwise divergences between samples were com-
prised between 2.9e-07 and 8.7e-07, corresponding to 0.00%.
Therefore, the WGS data confirmed that identical strains were recovered from two unre-
lated patients within the same hospital. In addition, the data indicate that the genomic charac-
terization following M13 typing allowed identifying and discriminating between strains.
At the same hospital (A), a B. cereus strain was isolated from a newborn (patient 4) and one
week later, a B. cereus strain with a similar profile was isolated from another premature new-
born with clinical sepsis (patient 3), constituting a second strain cluster.
Similarly, at the same hospital (A), a B. cereus strain was isolated from a premature newborn
with a clinical sepsis and brain abscesses (patient 2). A strain with similar profile was isolated
later twice from the hospital environment (environment 2 and 4), constituting a third cluster.
At hospital (B), a B. cereus strain was isolated from three newborns (patients 6, 8 and 9) and
from an environmental sample (environment 5) during the same month. Patient 6 had local B.
cereus colonization at the point of entry of catheter. The patient was treated with ceftriaxone
and the catheter was removed. Patient 8 had a contaminated gastric acid and patient 9 had a
bacteremia. They both received antibiotics including vancomycin and had favorable outcomes.
At hospital (E), strains with identical profiles were recovered from two patients over a one
year time interval and in different hospital wards (oncology and neurology). Patient 23 was
66-year old and diagnosed with colorectal cancer and sepsis. B. cereus was isolated from a
blood culture, displaying also a coagulase-negative Staphylococcus. B. cereus was therefore
neglected. However, a strain with identical profile was isolated one year later from a newborn
(patient 21) leading to kidney and urinary infections.
At the same hospital (E), B. cereus strains with similar profiles were recovered from three
different patients over a 2-year period. The three patients were hospitalized in different wards:
hematology, nephrology and gastroenterology. Patient 16, 65 year old had a positive blood cul-
ture and died without antibiotic treatment. Patient 17, age 54 had a bacteremia at the point of
entry of the catheter. He had a favorable outcome after several antibiotic courses for 21 days.
Patient 18, age 63, had three positive blood cultures yielding B. cereus strains with identical
profiles during three months. He had a favorable outcome following three consecutive antibi-
otic courses.
More recently, at hospital (F), B. cereus strains with identical profiles were isolated from
two patients over a one-year period. Patient 33 was 24 year-old admitted at the emergency
ward with abdominal pain, shivering, vomiting, fever and diarrhea. A blood culture was posi-
tive for this B. cereus strain. Patient 31, 48-year old, was admitted in cardiology almost one
year later with clinical sepsis and acute respiratory distress. A blood culture was positive with a
B. cereus with identical profile and the patient received a combined antibiotic course with a

favorable outcome. The two patients do not seem to have links in anyways and were admitted
at the hospital 10 months apart.
These data reveal the capacity of a given B. cereus strain to persist in the hospital and to infect
several patients over a long period of time (over 2 years) and at different hospital localizations.
Inter hospital contaminations. A case of inter-hospital contamination was identified
between the hospitals (A) and (B). A newborn from hospital (B) (patient 7) had a B. cereus
strain that presented an identical profile to a strain isolated the same month from the neonatal
hospital environment (environment 1) in hospital (A). As these data may reveal, to our knowl-
edge, the first inter hospital contamination ever described for B. cereus, we decided to compare
them as well by WGS. The identity between the two strains was confirmed, and the strains
showed 0.00% divergence and 20 differences in SNP (S4 Fig). No direct link has been identi-
fied between the patient 7 and hospital (A).
A second case of putative inter-hospital contamination was identified within the same hos-
pitals (A) and (B). At hospital (B), a B. cereus strain was isolated from three premature new-
borns (patients 6, 8 and 9) and from an environmental sample (environment 5) during the
same month. Surprisingly, a B. cereus strain with very similar profile (0.01% divergence and
48–49 differences in SNP) was isolated two months earlier from two newborns (patient 1 and
5) at hospital (A). Patients 1 and 5 from hospital (A) were the first newborns of this series. Dur-
ing the same period, patient 6 was first admitted in hospital (A) and then transferred to hospi-
tal (B), located 15 km apart. B. cereus strains with similar profiles were then isolated in hospital
(B) from patient 6, 8 and 9. According to the data, there is a possibility, that patient 6 may have
been contaminated during his stay in hospital (A) and transmitted the strain to other patients
in hospital (B).
Although it is not fully clear from the available evidence whether the isolates are indeed of
the same origin, these situations may be to our knowledge, the first examples of inter-hospital
cross-contaminations with B. cereus strains.
Discussion
B. cereus is notoriously associated with food poisoning and eye infections [31, 32]. B. cereus
also induces a multitude of other serious infections such as fulminant sepsis and devastating
central nervous system infections [9, 19]. In hospital, B. cereus is however usually regarded by
the physicians as an environmental contaminant. Thus, despite positive blood samples, B.
cereus is seldom considered as cause of infection. Consequently, the antibiotic treatment is
sometimes inadequate because of the misinterpretation of clinical and bacteriological diagno-
sis of B. cereus infections [33].
The aim of our study was to gain a better knowledge on the consequences, sources and
pathogenic strain patterns in B. cereus clinical infections. We analyzed the correlations
between epidemiology, clinical, phenotypical and molecular data in order to alert clinicians
regarding the emerging threat that B. cereus can represent in hospital settings.
We reported 39 patients with B. cereus infections. This study is however not exhaustive and
the number of cases is likely underestimated as clinical laboratories do not necessarily com-
plete species identification considering Bacillus species as environmental contaminants.
Among the 39 patients, eight (21%) died following B. cereus systemic infections. Among
them, 4 were premature newborns, 1 patient had a carcinoma, 1 patient was also infected with
S. aureus, 1 patient had a low leukocyte level. The underlying condition of the last one was
unknown. Consistent with previous findings [14, 18], our study confirms that the majority of
patients were premature newborns, followed by elderly people. It is suspected that B. cereus
from the hospital environment enter the infant bodies due to the presence of indwelling

devices such as catheters. However, to our knowledge, no studies could demonstrate so far that
the same B. cereus strain could be recovered from a patient and its hospital environment. The
comprehensive molecular characterization of the strains from our collection allowed identify-
ing several hospital clusters. Strains with identical profiles were isolated from different patients
and/or environment samples over periods of time up to two years and from different hospital
settings. This clearly suggests that the same B. cereus strain is able to persist in the hospital
environment despite routine cleaning procedures and may remain a source of infection for
inpatients, likely due to its ability to form spores and/or biofilms [31]. In addition, we reveal
the identification of strain clusters between two hospitals demonstrating the first documented
cases of inter-hospital cross-contamination by B. cereus strains. It is interesting to note that B.
cereus does not constitute a clonal population [34]. As an example, compared to the available
reference sequenced genomes B. thuringiensis 407 (https://www.ncbi.nlm.nih.gov/assembly/
GCF_000161495.1/ BT407) and B. cereus ATCC10987 (https://www.ncbi.nlm.nih.gov/
assembly/GCF_000008005.1/ ATCC10987), our strains showed 91.56% and 94.87% similarity,
respectively. In this situation, our WGS data showing 99.99% and 100% identity between the
hospital strains strongly suggest the strain identities.
In two cases, identical B. cereus strains were isolated from two newborns with different
pathologies. In both cases, the first infant had a localized colonization and the second had a
systemic infection, suggesting that the severity of symptoms probably depends on the site of
infection and/or the immune status of the patient.
Surface environmental samples were analyzed only in case of infection and were restricted
to the patient zone. The hands of the staff or the linen were not tested. It is therefore difficult
to hypothesize on the method of contamination. Our data, suggest however, that the strains
remain in the hospital environment long enough to infect inpatients up to two years following
the first infection. In two cases, the second infection led to patient death.
B. cereus gastrointestinal pathogenesis are considered to be mainly due to the production of
toxins such as HBL, NHE, CytK or the cereulide [28, 35–37]. The virulence factors associated
with clinical non-gastrointestinal diseases are unknown, although HlyII has been shown to
allow B. cereus to counteract the host immune system [38–40].
34% of the strains belonged to GS1 where only nhe genes were detected. Nevertheless, the
production of the NHE toxin was highly variable and ranged from high to very low, suggesting
that other factors may play a role during B. cereus non-gastro intestinal infections. High pro-
duction of one toxin NHE or HBL was correlated with low production of the other one.
Indeed, 54% of the strains were high NHE producers and low HBL producers, and only 25% of
the strains were high producers of both NHE and HBL. Thus, it appears necessary to identify
other unknown virulence determinants to get further insights in the pathogenic potential of
B. cereus during non-gastrointestinal infections or to establish whether such infections are
entirely opportunistic. It would be interesting to examine the role of the PlcR regulon, which is
suspected to play a major role during gastrointestinal diseases, as well as other non-PlcR regu-
lated toxins [35, 41, 42].
Interestingly, we observed that strains isolated from low age population were in average low
toxin-producers. This suggests that newborn may be particularly sensitive to B. cereus strains,
even those with low toxin production, or that other unknown factors may be responsible for
newborn infections.
There is no specific recommendation for the study and interpretation of B. cereus antibiotic
susceptibility in Europe. The choice of antibiotic is guided by therapeutic considerations and
the search for alternatives to the treatments used for prophylaxis. Our data show homogeneity
of antibiotic susceptibility pattern in the strain population, which is in favor of empiric therapy
as soon as B. cereus infection is identified. The data revealed the efficacy of the association of

glycopeptide and aminoglycoside or imipenem and ciprofloxacin. On the opposite, due to

the natural resistance of B. cereus to most beta-lactams [33] and as confirmed by our study,
penicillins and third generation cephalosporins are not recommend for treating B. cereus
infections.
Of interest, patient 18 had three blood samples positive for B. cereus. The initial strains
were susceptible to rifampicin and the last strain displayed resistance to rifampicin. This case
strongly suggests acquired rifampicin resistance over time, and that, similarly to S. aureus,
rifampicin should be used with caution to treat B. cereus infections. Whether the strain
acquired resistance from another bacterium or from a mutation was not studied.
Taken together, such study gathering epidemiological and clinical data together with phe-
notypic and molecular characterization has, to the best of our knowledge, never been done
for B. cereus. This study demonstrates the high persistence capacity of B. cereus strains in the
hospital environment, leading to the reemergence of strains two years after the first isolation.
Strains spread within the same hospital but also between different hospitals.
The antibiotic resistance profiles should allow to quickly adapting treatment and care of
patients. In conclusion, our study highlights that B. cereus isolated from patients, especially
if immunosuppressed, should not be systematically disregarded as a contaminant, and its clini-
cal significance should be raised. Inadequate attention could delay appropriate therapy and
increase the risk of severe infections and poor outcome.
Supporting information
S1 Fig. Primers. List of primers used in this study.
(TIFF)
S2 Fig. Toxin gene profiles called genetic signatures (GS). The toxin gene profiling was per-
formed according to the presence or absence of nine genes (cytK1 was absent in all strains)
associated with B. cereus pathogenesis.
(TIFF)
S3 Fig. In vitro antibiotic susceptibility profiles. Five profiles were defined from the 56 B.
cereus isolated from patients or from hospital environment. S: susceptible R: resistant.
(TIFF)
S4 Fig. SNP calling and pairwise divergence. SNP calling and pairwise divergence were calcu-
lated between the samples.
(TIFF)
Acknowledgments
We wish to thank Joel Grout, Sylvie Pairaud, Muriel Marault and Sabine Messio for excellent
technical assistance. We thank Stéphane Aymerich for his kind support. We thank Pierre
Nicolas, Jacques Croizé and Jacqueline Tous for helpful discussion. We are grateful to the
INRA MIGALE bioinformatics platform (http://migale.jouy.inra.fr) for providing computa-
tional resources.
Author Contributions
Conceptualization: Benjamin Glasset, Sabine Herbin, Sophie A. Granier, Anne Brisabois,
Nalini Ramarao.
Data curation: Nalini Ramarao.

Formal analysis: Benjamin Glasset, Sabine Herbin, Cyprien Guérin, Anne Brisabois, Nalini
Ramarao.
Funding acquisition: Anne Brisabois, Nalini Ramarao.
Investigation: Nalini Ramarao.
Methodology: Benjamin Glasset, Sabine Herbin, Sophie A. Granier, Laurent Cavalié, Emilie
Lafeuille, Cyprien Guérin, Raymond Ruimy, Florence Casagrande-Magne, Marion Levast,
Nathalie Chautemps, Jean-Winoc Decousser, Laure Belotti, Isabelle Pelloux, Jerôme Robert,
Anne Brisabois, Nalini Ramarao.
Project administration: Nalini Ramarao.
Resources: Sabine Herbin, Laurent Cavalié, Emilie Lafeuille, Raymond Ruimy, Florence Casa-
grande-Magne, Marion Levast, Nathalie Chautemps, Jean-Winoc Decousser, Laure Belotti,
Isabelle Pelloux, Jerôme Robert, Anne Brisabois, Nalini Ramarao.
Software: Sabine Herbin, Cyprien Guérin, Nalini Ramarao.
Supervision: Sabine Herbin, Sophie A. Granier, Anne Brisabois, Nalini Ramarao.
Validation: Benjamin Glasset, Raymond Ruimy, Nalini Ramarao.
Visualization: Nalini Ramarao.
Writing – original draft: Benjamin Glasset, Nalini Ramarao.
Writing – review & editing: Sabine Herbin, Jerôme Robert, Anne Brisabois, Nalini Ramarao.
References
1. Auger S, Ramarao N, Faille C, Fouet A, Aymerich S, Gohar M. Biofilm formation and cell surfce proper-
ties among pathogenic and non pathogenic strains of the Bacillus cereus group. App Environ Microbiol.
2009; 75:6616–8.
2. Tran SL, Guillemet E, Gohar M, Lereclus D, Ramarao N. CwpFM (EntFM) is a Bacillus cereus potential
cell wall peptidase implicated in adhesion, biofilm formation and virulence. J Bacteriol. 2010; 192:2638–
42. https://doi.org/10.1128/JB.01315-09 PMID: 20233921
3. Glasset B, Herbin S, Guiller L, Cadel-Six S, Vignaud ML, Grout J, et al. Large-scale survey of Bacillus
cereus-induced food-borne outbreaks: epidemiologic and genetic characterization EuroSurveillance.
2016; 21(48).
4. INVS. Collective Food Poisoning INVS. 2016.
5. Veysseyre F, Fourcade C, Lavigne JP, Sotto A. Bacillus cereus infection: 57 case patients and a litera-
ture review. Med Mal Infect. 2015; 45(11–12):436–40. https://doi.org/10.1016/j.medmal.2015.09.011
PMID: 26525185
6. Ramarao N. Bacillus cereus: caractéristiques et pathogénicité. EMC Biologie Médicale. 2012; 7:1–11.
7. Kato K, Matsumura Y, Yamamoto M, Nagao M, Ito Y, Takakura S, et al. Erratum to: Seasonal trend and
clinical presentation of Bacillus cereus bloodstream infection: association with summer and indwelling
catheter. Eur J Clin Microbiol Infect Dis. 2016; 35(5):875–83. https://doi.org/10.1007/s10096-016-2618-
8 PMID: 27010814
8. Frankard J, Li R, Taccone F, Struelens M, J, Jacobs F, Kentos A. Bacillus cereus pneumonia in a
patient with acute lymphoblastic leukemia. Eur J Clin Microbiol Infect Dis. 2004; 23:725–8. https://doi.
org/10.1007/s10096-004-1180-y PMID: 15300457
9. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 2010; 23(2):382–98.
https://doi.org/10.1128/CMR.00073-09 PMID: 20375358
10. Gaur AH, Patrick CC, McCullers JA, Flynn PM, Pearson TA, Razzouk BI, et al. Bacillus cereus bacter-
emia and meningitis in immunocompromised children. Clinic Infect dis. 2001; 32:1456–62.
11. Arnaout M, Tamburro R, Bodner S, Sandlund J, Rivera G, Pui C. Bacillus cereus causing fulminant sep-
sis and hemolysis in two patients with acute leukemia. J Pediatr Hematol Oncol. 1999; 21:431–5. PMID:
10524460

12. Wright WF. Central Venous Access Device-related Bacillus Cereus Endocarditis: A Case Report and
Review of the Literature. Clin Med Res. 2016.
13. Shah M, Patnaik S, Wongrakpanich S, Alhamshari Y, Alnabelsi T. Infective endocarditis due to Bacillus
cereus in a pregnant female: A case report and literature review. IDCases. 2015; 2(4):120–3. https://
doi.org/10.1016/j.idcr.2015.10.003 PMID: 26793477
14. Lotte R, Herisse AL, Berrouane Y, Lotte L, Casagrande F, Landraud L, et al. Virulence Analysis of Bacil-
lus cereus Isolated after Death of Preterm Neonates, Nice, France, 2013. Emerg Infect Dis. 2017; 23
(5):845–8. https://doi.org/10.3201/eid2305.161788 PMID: 28418291
15. Evreux F, Delaporte B, Leret N, Buffet-Janvresse C, Morel A. [A case of fatal neonatal Bacillus cereus
meningitis]. Arch Pediatr. 2007; 14(4):365–8. https://doi.org/10.1016/j.arcped.2007.01.009 PMID:
17337168
16. Hilliard NJ, Schelonka RL, Waites KB. Bacillus cereus bacteremia in a preterm neonate. J Clin Micro-
biol. 2003; 41(7):3441–44. https://doi.org/10.1128/JCM.41.7.3441-3444.2003 PMID: 12843116
17. Decousser J, Ramarao N, Duport C, Dorval M, Bourgeois-Nicolaos N, Guinebretiere MH, et al. Bacillus
cereus and severe intestinal infections in preterm neonates: putative role of the pooled breast milk. Am
Journal of Infection Control. 2013; 41:918–21.
18. Ramarao N, Belotti L, Deboscker S, Ennahar-Vuillemin M, de Launay J, Lavigne T, et al. Two unrelated
episodes of Bacillus cereus bacteremia in a neonatal intensive care unit. Am J Infect Control. 2014; 42
(6):694–5. https://doi.org/10.1016/j.ajic.2014.01.025 PMID: 24725514
19. Drobniewski FA. Bacillus cereus and related species. Clin Microbiol Rev. 1993; 6:324–38. PMID: 8269390
20. Miller JM, Hair JG, Hebert M, Hebert L, Roberts FJ, Weyant RS. Fulminating bacteremia and pneumo-
nia due to Bacillus cereus. J Clin Microbiol. 1997; 35(2):504–7. PMID: 9003628
21. Hoffmaster A, Ravel J, Rasko D, Chapman GD C M, Marston CK, De BK, Sacchi CT, Fitzgerald C,
Mayer LW, Maiden MC, Priest FG, Barker M, Jiang L, Cer RZ, Rilstone J, Peterson SN, Weyant RS,
Galloway DR, Read TD, Popovic T, Fraser CM. Identification of anthrax toxin genes in a Bacillus cereus
associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci. 2004; 101:8449–54.
https://doi.org/10.1073/pnas.0402414101 PMID: 15155910
22. Marston CK, Ibrahim H, Lee P, Churchwell G, Gumke M, Stanek D, et al. Anthrax Toxin-Expressing
Bacillus cereus Isolated from an Anthrax-Like Eschar. PLoS ONE. 2016; 11(6):e0156987. https://doi.
org/10.1371/journal.pone.0156987 PMID: 27257909
23. Benusic MA, Press NM, Hoang LM, Romney MG. A cluster of Bacillus cereus bacteremia cases among
injection drug users. Can J Infect Dis Med Microbiol. 2015; 26(2):103–4. PMID: 26015795
24. Sasahara T, Hayashi S, Morisawa Y, Sakihama T, Yoshimura A, Hirai Y. Bacillus cereus bacteremia
outbreak due to contaminated hospital linens. Eur J Clin Microbiol Infect Dis. 2011; 30(2):219–26.
https://doi.org/10.1007/s10096-010-1072-2 PMID: 20938704
25. Beecher DJ, Wong AC. Identification of hemolysin BL-producing Bacillus cereus isolates by a discontin-
uous hemolytic pattern in blood agar. Appl Environ Microbiol. 1994; 60(5):1646–51. PMID: 8017944
26. Bouillaut L, Ramarao N, Buisson C, Gilois N, Gohar M, Lereclus D, et al. FlhA influences Bacillus thurin-
giensis PlcR-regulated gene transcription, protein production, and virulence. Appl Environ Microbiol.
2005; 71(12):8903–10. https://doi.org/10.1128/AEM.71.12.8903-8910.2005 PMID: 16332888
27. Guinebretière MH, Thompson FL, Sorokin A, Normand P., Dawyndt P., Ehling-Schulz M, et al. Ecologi-
cal diversification in the Bacillus cereus Group. Environ Microbiol. 2008; 10:851–65. https://doi.org/10.
1111/j.1462-2920.2007.01495.x PMID: 18036180
28. Guinebretière MH, Broussolle V, Nguyen-The C. Enterotoxigenic profiles of food-poisoning and food-
borne Bacillus cereus strains. J Clin Microbiol. 2002; 40(8):3053–6. https://doi.org/10.1128/JCM.40.8.
3053-3056.2002 PMID: 12149378
29. Wayne. Clinical and Laboratory Standards Institute (CLSI). Methods for antimicrobial dilution and disk
susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline. CLSI. 2010; 2nd
edition: M45A2E.
30. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of
Simpson’s index of diversity. J Clin Microbiol. 1988; 26(11):2465–6. PMID: 3069867
31. Ramarao N, Lereclus D, Sorokin A. The Bacillus cereus group. Molecular Medical Microbiology. 2015;
III(Second Edition):1041–78.
32. Callegan MC, Kane ST, Cochran DC, Novosad B, Gilmore MS, Gominet M, et al. Bacillus endophthal-
mitis: roles of bacterial toxins and motility during infection. Invest Ophthalmol Vis Sci. 2005; 46
(9):3233–8. https://doi.org/10.1167/iovs.05-0410 PMID: 16123424
33. Ikeda M, Yagihara Y, Tatsuno K, Okazaki M, Okugawa S, Moriya K. Clinical characteristics and antimi-
crobial susceptibility of Bacillus cereus blood stream infections. Ann Clin Microbiol Antimicrob. 2015;
14:43. https://doi.org/10.1186/s12941-015-0104-2 PMID: 26370137

34. Rasko D A, Ravel J, Okstad OA, Helgason E, Cer R, Z, Jiang L, et al. The genome sequence of Bacillus
cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis
pXO1. Nucleic Acids Res. 2004; 32:977–88. https://doi.org/10.1093/nar/gkh258 PMID: 14960714
35. Ramarao N, Sanchis V. The pore-forming haemolysins of Bacillus cereus: a review. Toxins. 2013;
5:1119–39. https://doi.org/10.3390/toxins5061119 PMID: 23748204
36. Stenfors Arnesen L, Fagerlund A, Granum P. From soil to gut: Bacillus cereus and its food poisoning
toxins. FEMS Microbiol Rev. 2008; 32:579–606. https://doi.org/10.1111/j.1574-6976.2008.00112.x
PMID: 18422617
37. Ehling-Schulz M, Guinebretière MH, Monthan A, Berge O, Fricker M, Svensson B. Toxin gene profiling
of enterotoxic and emetic Bacillus cereus. FEMS Microbiol Lett. 2006; 260:232–40. https://doi.org/10.
1111/j.1574-6968.2006.00320.x PMID: 16842349
38. Tran SL, Guillemet E, Ngo-Camus M, Clybouw C, Puhar A, Moris A, et al. Hemolysin II is a Bacillus
cereus virulence factor that induces apoptosis of macrophages. Cell Microbiol. 2011; 13:92–108.
https://doi.org/10.1111/j.1462-5822.2010.01522.x PMID: 20731668
39. Tran SL, Puhar A, Ngo-Camus M, Ramarao N. Trypan blue dye enters viable cells incubated with the
pore-forming toxin HlyII of Bacillus cereus. PLoS ONE. 2011; 6(9):e22876. https://doi.org/10.1371/
journal.pone.0022876 PMID: 21909398
40. Guillemet E, Tran S, Cadot C, Rognan D, Lereclus D, Ramarao N. Glucose 6P binds and activates
HlyIIR to repress Bacillus cereus haemolysin hlyII gene expression. PLoS ONE. 2013; 8:e55085.
https://doi.org/10.1371/journal.pone.0055085 PMID: 23405113
41. Gohar M, Faegri K, Perchat S, Ravnum S, Økstad O G M.; Kolstø AB.; Lereclus D. The PlcR virulence
regulon of Bacillus cereus. PLoS ONE. 2008; 3(7):e2793. https://doi.org/10.1371/journal.pone.0002793
PMID: 18665214
42. Salamitou S, Ramisse F, Brehélin M, Bourguet D, Gilois N, Gominet M, et al. The plcR regulon is
involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects.
Microbiology. 2000; 146:2825–32. https://doi.org/10.1099/00221287-146-11-2825 PMID: 11065361

Cereus 3

Uploaded by

Copyright:

Available Formats

Cereus 3

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Cereus 3

Uploaded by

Copyright:

Available Formats

RESEARCH ARTICLE

Bacillus cereus, a serious cause of nosocomial

Funding: The authors received no specific funding

Competing interests: The authors have declared

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 1 / 19

Material and methods

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 2 / 19

Genomic divergence estimation

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 3 / 19

ampicillin$, cefotaxime, imipenem$, vancomycin$, gentamicin$, rifampicin$, tetracycline$, cip-

Molecular typing and statistical analysis

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 4 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 5 / 19

Table 1. Epidemiological and clinical data of patients and samplings.

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 6 / 19

Table 2. Characteristics of patients or hospital environment displaying B. cereus positive samples.

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 7 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 8 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 9 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 10 / 19

Principal component analyses

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 11 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 12 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 13 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 14 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 15 / 19

glycopeptide and aminoglycoside or imipenem and ciprofloxacin. On the opposite, due to

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 16 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 17 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 18 / 19

PLOS ONE | https://doi.org/10.1371/journal.pone.0194346 May 23, 2018 19 / 19

You might also like