ORIGINAL ARTICLE

Vigorous cleaning and adequate ventilation are necessaryto control an outbreak in a neonatal intensive care unit

Nobuyuki Shimono • Jun Hayashi • Hiroko Matsumoto •

Noriko Miyake • Yujiro Uchida • Shinji Shimoda •

Norihiro Furusyo • Koichi Akashi

Received: 6 January 2011 / Accepted: 2 October 2011 / Published online: 26 October 2011

� Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2011

Abstract An outbreak of Bacillus cereus (B. cereus)

bacteremia occurred in our neonatal intensive care unit

(NICU) in July 2005. Many strains of B. cereus were

cultured from patient specimens, as well as from envi-

ronmental samples such as the surfaces of instruments and

air in the NICU. Some of these strains were analyzed by

pulsed field gel electrophoresis, and several were con-

firmed to be identical. We speculated that the bacterial

load in the environment had initially increased and then

possibly spread throughout the NICU facility via the air-

flow of the ventilation system. For this reason, besides

maintaining standard precautions, we performed a vigor-

ous clean of the NICU, and covered the vents to prevent

dust falling from them. These protective measures ended

the outbreak. In the hospital environment, adequate

ventilation is important, especially in single-occupancy

isolation rooms and operating theaters. However, the

criteria for the adequate ventilation of multioccupancy

rooms for acute care environments such as the NICU have

not yet been defined. We need to pay more attention to

these environmental factors in order to avoid cross con-

tamination and infectious outbreaks.

Keywords Bacillus cereus � Ventilation �Multioccupancy rooms � Hospital infection outbreak

Introduction

Bacillus cereus (B. cereus) is a Gram-positive rod that

can cause food poisoning. B. cereus forms endospores,

making it resistant to heat, detergents and alcohol. This

characteristic allows B. cereus to exist in a wide variety

of environments. When B. cereus is isolated from clin-

ical samples, it is difficult to confirm whether it is a

cause of infectious diseases or simply a contaminant.

Even if B. cereus is isolated from blood cultures, the

results should usually be reconfirmed. However, it is also

true that once B. cereus causes bacteremia, the patient’s

condition can become serious. Serial isolation of

B. cereus in blood cultures was noticed in July of 2005

in the neonatal intensive care unit (NICU) of our

hospital.

Most nosocomial infections are thought to be transmit-

ted via the direct contact route. However, despite relatively

good adherence to standard and contact precautions, we

experienced the aforementioned outbreak, raising the

possibility that the airborne route might play an important

role in nosocomial infections. Besides strains from blood

cultures, we collected samples from various items in the

NICU, ventilation grills and air samples. As a result, we

N. Shimono (&) � J. Hayashi � H. Matsumoto � N. Miyake �Y. Uchida � N. Furusyo

Department of Infection Control and Prevention/Infection

Control Team, Kyushu University Hospital, 3-1-1 Maidashi,

Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan

e-mail: shimono@intmed1.med.kyushu-u.ac.jp

N. Shimono � N. Miyake � Y. Uchida

Department of Clinical Immunology

and Rheumatology/Infectious Diseases,

Kyushu University Hospital, Fukuoka, Japan

N. Shimono � N. Miyake � Y. Uchida � S. Shimoda � K. Akashi

Department of Medicine and Biosystemic Science,

Kyushu University Graduate School of Medical Sciences,

Fukuoka, Japan

J. Hayashi � N. Furusyo

Department of General Medicine, Kyushu University Hospital,

Fukuoka, Japan

123

J Infect Chemother (2012) 18:303–307

DOI 10.1007/s10156-011-0326-y

underscore the importance of ventilation in multioccu-

pancy rooms like the NICU.

Patients and methods

Clinical setting and investigation

Kyushu University Hospital is a 1,300-bed tertiary care

hospital in Fukuoka, Japan. Our NICU had 21 beds and

provided a high standard of medical care for neonates. At

the time of the outbreak, the NICU was divided into two

rooms (NICU 1 and NICU 2), but there was no door

between them. The ventilation system had inlets and vents

for air in the NICU, which were located on the ceiling.

Exhaust air was filtered and mixed with fresh air from

outside the building. Finally, air was supplied to the NICU

via the inlets after passing through high-efficiency partic-

ulate air (HEPA) filters (Fig. 1a).

Clinical specimens from the nasal cavity, throat, spu-

tum, urine, stool and other sites of infection were cultured

as needed. Medical charts and microbiological records

were reviewed.

Environmental cultures

At the beginning of August 2005, specimens were obtained

from items in the NICU, including several types of anti-

septics, ethanol, tap water, cotton-tipped swabs, cotton

balls, and so on. A total of 20 specimens were inoculated

onto 5% sheep blood agar and BTB (bromothymol blue)

agar plates.

A total of 28 samples were collected from table surfaces,

inside and outside of infant incubators, access port covers,

and ventilation grills, using soaked cotton swabs.

Linen pieces (each 2 cm2) were soaked in sterile water

in 50 ml plastic tubes. The tubes were vortex-mixed, cen-

trifuged, and the pellets were finally cultured in the same

manner as described above.

Samples were taken from the bare hands of ten staff

members before and after washing with soap, and after

wearing gloves. They were cultured by the stamp method

on blood agar plates.

Air samples were collected using an air sampler

(AirTrend, Kuraray Co., Ltd., Okayama, Japan) that was

set to collect 6.29 L/min for 18 min. These samples were

collected in the corner of NICU and in control spaces

(outside the NICU). After air sample collection (four in

NICU1 and four in NICU2), the cartridges containing

agar were incubated at 37�C overnight. Colonies were

counted and each colony was identified under micro-

scope after Gram staining. The strains were identified as

B. cereus after re-culturing on mannitol egg yolk

polymyxin (MYP) agar plates (Becton, Dickinson and

Co., Franklin Lakes, NJ, USA). Lack of mannitol fer-

mentation and presence of lecithinase production were

suspected and confirmed by an API 50CHB/API 20E

system (bioMerieux, Lyon, France).

Molecular analysis

Pulsed-field gel electrophoresis (PFGE; Bio-Rad Labora-

tories, Richmond, CA, USA) was performed on available

isolates obtained from patients and environmental speci-

mens. Genomic DNAs were prepared as described in the

instruction manual and application guide provided by

Bio-Rad Laboratories. DNAs were restricted using the

enzyme SmaI, electrophoresed on a CHEF-DRII system

(Bio-Rad Laboratories), then photographed, and restriction

profiles were finally compared visually.

filterHEPAfilter

fresh air

Doorturbulance

inletvent

Room 1 Room 2

Vent grill Vent grill

cover

Before After

Air Flow

Dust

a

b

Fig. 1 Diagram of the ventilation system and a cover installed

beneath the vent. a Diagram of the ventilation system in the NICU.

Exhaust air from the vent was filtered and mixed with fresh air from

outside the building. Finally, HEPA-filtered air was re-supplied

through the inlet to the NICU. b Diagram of a cover installed beneath

the vent. Cotton-like dust was present on the vent grill, though the

inlet grill was clean. The cover prevented dust from falling from the

vent grill

304 J Infect Chemother (2012) 18:303–307

123

Results

Clinical investigation

Our infection control team (ICT) had not routinely sur-

veyed for all types of bacteria, but the NICU staff had

noticed the frequent isolation of B. cereus from clinical

specimens at the end of July 2005. They reported this to

the ICT at the beginning of August, and confirmed an

increase in the monthly positive isolation rate (positive/

total specimens). The isolation rate between April and

July was 7.5–9.6%, compared with the baseline rate of

3.2–4.7%. We thus conducted rounds, reviewed micro-

biological records, and collected environmental speci-

mens. According to these records, B. cereus strains were

isolated from five, six, four, and six patients in April,

May, June, and July, respectively. We had 21 infants in

the NICU at the end of July, and B. cereus had been

isolated from 16 of them (76.2%) at some point during

the time of this study. Moreover, in July, three patients

had blood cultures positive for B. cereus. All three

patients presented clinical manifestations of infection,

such as fever and elevation of inflammatory values.

Gestational age, birth weight, age in days, and the

locations of these patients were as follows: patient A,

29 weeks, 476 g, 2 day, incubator; patient B, 30 weeks,

876 g, 4 day, incubator; patient C, 28 weeks, 1,018 g,

3 day, incubator. All three patients recovered due to the

administration of antibiotics.

Environmental cultures

We first suspected that the increased isolation rate of

B. cereus would be attributable to the contamination of items

in the NICU. However, unexpectedly, cultures from all items

and liquid specimens from the NICU yielded no organisms

(data not shown). Therefore, we collected samples from the

surfaces of objects in the environment using soaked swabs.

Hands were cultured by the stamp method using blood agar

plates. Ventilation vents and inlets were located on the

ceiling in the NICU, and a table was located just under the

vent. Numerous B. cereus colonies grew from all eight

specimens from the vent grills and five specimens from the

table, while none grew from those from the inlet grills.

Moderate or greater numbers of colonies were recovered

from the specimens from unwashed and gloved hands of

seven staff members. Small numbers of colonies were also

recovered from the surfaces of infant incubators; even

their internal surfaces. We suspected contamination with

B. cereus in the NICU itself, which we attempted to confirm

by air sampling. Seven to ten colonies grew from each air

sample, and the majority of these colonies from the NICU

were identified as B. cereus, while no B. cereus colonies grew

from the control air samples.

Molecular analysis

Figure 2 depicts the PFGE patterns of genomic DNA after

SmaI endonuclease digestion of all available B. cereus

M 1 2 3 4 5 6 7 8 9 101112 13 14 1516 17 1819 20 21 222324 25 26 Mkb

145 5

194.0242.5291.0

48

97.0

145.5

48.5

Fig. 2 Pulsed-field gel electrophoresis (PFGE) profiles of B. cereusisolates. M molecular weight marker (k ladder, Bio-Rad). Isolates:

lane 1 blood (patient A, July 15); lane 2 blood (patient B, July 30);

lane 3 blood (patient C, July 31); lane 4 incubator (patient D, August

14); lane 5 nasal cavity (patient E, July 18); lane 6 incubator access

port cover (patient D, August 7); lane 7 cart surface (room 1, August

14); lane 8 unused glove (August 7); lane 9 center table (room 1,

August 14); lane 10 vent grill (room 2, August 23); lane 11bronchoalveolar lavage fluid (BAL) (patient F, August 7); lane 12

nasal cavity (patient G, August 5); lane 13 staff member’s hand

(August 7); lane 14 echo panel surface (August 23); lane 15 nasal

cavity (patient H, September 10); lane 16 vent grill (room 1,

September 12); lane 17, 18 air (room 1, September 12); lane 19–21air (room 2, September 12); lane 22 vent grill (room 2, September

12); 23–25 vent grill (room 1, September 12); 26 S. aureus as a

control. Lanes with the same symbols are those which seemed to be

identical

J Infect Chemother (2012) 18:303–307 305

123

isolates from clinical and environmental samples. Marked

isolates were considered to be identical. For example,

strains from blood cultures of patient A and patient B were

identical. Strains from the blood culture of patient C and

strains from the infant incubator of patient D were also

identical.

Cleaning the room and installing covers beneath

the ventilation vents

In October, we installed covers beneath the vents to pre-

vent dust from falling from the vent grills. Besides per-

forming adequate maintenance of the HEPA filters fitted to

the inlets, we also decided to start cleaning the vent grills

every four months. We also started to clean the room,

tables, and so on vigorously. The isolation rate of B. cereus

from clinical samples gradually decreased to 2.2–2.8%

after these measures had been instituted. There were also

no bacteremia patients six months after intervention. When

air sampling was performed after the intervention, 4–6

colonies grew from each sample, and 1–2 were identified as

B. cereus.

Discussion

The isolation rate of B. cereus in clinical NICU specimens

increased from April through July 2005. B. cereus is

widely distributed in the environment, but is rarely of

clinical significance because its pathogenicity is low. Even

when B. cereus is isolated from blood cultures, it is usually

thought to be a contaminant. In the present investigation,

we confirmed bloodstream infections based on clinical

symptoms and blood test results. Three bloodstream

infections constituted an outbreak.

We launched this investigation by initally collecting

samples from various items, environmental surfaces, and

the gloved and bare hands of NICU staff. No B. cereus was

isolated from any equipment in the NICU, but we con-

firmed that B. cereus was present on various environmental

surfaces and on both the bare and gloved hands of staff

members.

We also collected air samples using an air sampler, and

significant B. cereus colonies were isolated from NICU air

samples. We performed PFGE, which showed that while

strains isolated from clinical and environmental samples

were not all identical, some were the same, and they could

be divided into definite groups. This indicated that several

strains of B. cereus had spread widely and increased the

bacterial load of B. cereus inside the NICU. This increase

in the quantity of these bacteria ultimately might have

caused bloodstream infections.

Several B. cereus outbreaks caused by contamination of

linens have been reported [1]. This is attributable to the

formation of endospores by B. cereus, making it resistant to

heat, disinfectants, and alcohol. Severe contamination of a

washing machine with B. cereus can lead to the contami-

nation of linens and nosocomial infection outbreaks. In our

case, we did not isolate B. cereus from linens, nor find a

common source of nosocomial infection.

The main transmission route for nosocomial infection is

usually direct contact. Thus, adherence to standard pre-

cautions, especially vigorous hand washing, is important

for the prevention of nosocomial infection. Though hand

washing is undoubtedly essential to prevent the spread of

methicillin-resistant Staphylococcus aureus (MRSA)

infection, some researchers insist that the airborne route

should also be considered [2–4]. In other words, bacteria

that are resistant to desiccation, such as MRSA and

Acinetobacter species, are thought to spread and be trans-

mitted via the airborne route in addition to contact trans-

mission [5]. Taking into consideration that B. cereus forms

endospores and is more resistant to desiccation than

MRSA, B. cereus might be transmitted by the same route,

allowing it to exist in the hospital environment for a long

time and widely contaminate a relatively enclosed envi-

ronment such as the NICU.

The ventilation system of the hospital is important for

the prevention of airborne infection [6]. For single rooms

on the bone marrow transplantation (BMT) ward espe-

cially, as well as for isolation rooms and operating theaters

[7, 8], an adequate ventilation system is important for

preventing nosocomial airborne infections such as asper-

gillosis, legionellosis, tuberculosis, measles, and so on. We

experienced a dramatic decrease in the number of asper-

gillosis patients when we used a new BMT ward equipped

with HEPA filters and employed positive pressure versus

the corridor [9]. For a single room in the BMT ward and in

the operating theater, it is important to increase the number

of ventilations and to maintain positive pressure versus the

corridor to prevent cross contamination. It is also important

to increase the number of ventilations while maintaining

negative pressure in isolation rooms.

The NICU is where newborn babies receive intensive

care. These neonates are considered to be immune-com-

promised hosts. For adequate infection management, staff

members must wash their hands thoroughly and wear

gloves when touching babies. However, the NICU is a so-

called multioccupancy room; i.e., there are multiple incu-

bators, open incubators, and cots in the same room. Inside

the incubator, a neonate is protected from infection, but the

neonate is not fully protected in a cot or when the incubator

is opened. Another problem is that there is no single room

in the NICU, and the distance between beds is not

sufficient.

306 J Infect Chemother (2012) 18:303–307

123

The NICU is a quasi-clean area, with its cleanliness

level being equivalent to class 7 of the International

Organization for Standardization (ISO) standard, or class

10,000 of the NASA bio-clean room standard applied in

our hospital. In order to maintain this high level of clean-

liness, the ventilation program is set to increase the

ventilation volume and number in the NICU. However, it is

important to note that this cleanliness level is assured only

when staff members do not go in and out, and the door

between the NICU and adjacent corridor is closed. How-

ever, numerous people go in and out, including NICU

personnel and patients’ family members, and the door

between the NICU and corridor is usually open, making it

impossible to maintain an adequately high level of

cleanliness.

In our NICU, ventilation vents and inlets are both on the

ceiling, and massive ventilation was employed to maintain

the cleanliness level. This mixing of air and massive ven-

tilation is effective at eliminating pathogens such as viruses

via air exhaust (Fig. 1a) [10]. However, under the condi-

tions described above, with movement of visitors and

personnel, excess ventilation would cause turbulence,

possibly leading to the spread of pathogens (for example

B. cereus) in the environment. In order to minimize tur-

bulence, we decided to renovate our NICU in order to

change the airflow from the mixing ventilation pattern to a

unidirectional pattern. In other words, we wanted to place

vents at the lower corners of the room. However, this

renovation was found to be impossible because of financial

restrictions. Instead, we installed covers beneath the vents

to prevent dust falling from the vent grill on the ceiling in

October (Fig. 1b). We also started to clean the vent grills

periodically (every 4 months) and maintained a clean

NICU, thereby decreasing the bacterial load. These inter-

vention measures decreased the isolation rate of B. cereus

in clinical samples, and the outbreak ended.

There are several reports and guidelines on ventilation

systems for single rooms in BMT wards, isolation rooms

for infectious patients, operating theaters, and so on [6, 11].

In contrast, only a few reports have focused on multioc-

cupancy rooms [10] or wards, and there are no precise

guidelines. For ward ventilation, in general, the only

requirements are to provide patients with a comfortable

temperature and humidity. Generally, little attention is paid

to avoiding cross contamination. Herein, we emphasize the

need to investigate optimal ventilation systems that are

designed to avoid nosocomial infections in multioccupancy

rooms, especially where compromised hosts are cared for,

such as in the NICU.

Acknowledgments We thank Ms. Kazuko Hosokawa and

Ms. Mikiko Ushijima for their excellent technical assistance.

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