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Significance of Airborne Transmission of Methicillin-Resistant Staphylococcus aureus in an Otolaryngology–Head and Neck Surgery Unit
Teruo Shiomori, MD, PhD;
Hiroshi Miyamoto, MD, PhD;
Kazumi Makishima, MD, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:644-648.
ABSTRACT
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Objectives To quantitatively investigate the existence of airborne methicillin-resistant Staphylococcus aureus (MRSA) in a hospital environment
and to perform phenotyping and genotyping of MRSA isolates to study MRSA epidemiology.
Design Prospective surveillance of patients with MRSA infections or colonizations
was performed, as was an observational study of environmental airAirborne
samples were taken by an air sampler; samples were obtained from object surfaces
by stamping or swabbing. Epidemiological study of MRSA isolates was performed
with an antibiotic susceptibility test, coagulase typing, and pulsed-field
gel electrophoresis.
Setting Three single-patient rooms in a 37-bed otolaryngology–head and
neck surgery unit.
Patients Three patients with squamous cell head and neck cancer were observed
to have been colonized or infected with MRSA after surgery.
Results The MRSA samples were collected from the air in single-patient rooms
during both a period of rest and when bedsheets were being changed. Isolates
of MRSA were detected in all stages (from stage 1 [>7 µm] to stage 6
[0.65-1.1 µm]). About 20% of the MRSA particles were within a respirable
range of less than 4 µm. Methicillin-resistant S aureus was also isolated from inanimate environments, such as sinks, floors,
and bedsheets, in the rooms of the patients with MRSA infections as well as
from the patients' hands. An epidemiological study demonstrated that clinical
isolates of MRSA in our ward were of one origin and that the isolates from
the air and from inanimate environments were identical to the MRSA strains
that caused infection or colonization in the inpatients.
Conclusions Methicillin-resistant S aureus was recirculated
among the patients, the air, and the inamimate environments, especially when
there was movement in the rooms. Airborne MRSA may play a role in MRSA colonization
in the nasal cavity or in respiratory tract MRSA infections. Measures should
be taken to prevent the spread of airborne MRSA to control nosocomial MRSA
infection in hospitals.
INTRODUCTION
STAPHYLOCOCCUS aureus
is a common pathogen observed in the head and neck region. Antibiotic treatment
of most infections in the head and neck region must take into account the
prevalence of this organism because S aureus is also
one of the most common causes of nosocomial infections. During the past 20
years, methicillin-resistant S aureus (MRSA) has
become an important source of such infections; this pathogen is presently
responsible for up to 61% of Staphylococcus infections.1 Many patients in the otolaryngology unit have chronic
or recurrent infections, such as chronic otitis media or sinusitis. Such patients
may be prone to an increased rate of MRSA infections as the result of repeated
antibiotic therapy.1 Furthermore, patients
who have undergone head and/or neck operations, such as middle ear surgery,
usually have long-term packing in their auditory canals or mastoid cavities;
the packed gauzes are kept wet by transudates or exudates,2
and these materials are susceptible to contamination by pathogenic bacteria
such as MRSA. Therefore, otolaryngology–head and neck surgery units
are especially vulnerable to the spread of MRSA infections.3
The principal mode of MRSA transmission within an institution is from
patient to patient via transiently colonized hands of hospital personnel who
acquire the organism after direct patient contact or after handling contaminated
materials.4 Since MRSA isolates have been recovered
from many sites, including floors, linens, medical equipment, and hospital
furnishings, transmission via inanimate environments may also pose significant
risk to the patient.4 In addition, it is thought
that MRSA in the form of a bioaerosol can contaminate the air and cause airborne
infectious diseases.5 Although airborne transmission
of MRSA is generally considered to be less frequent than transmission via
direct contact, airborne MRSA is an important factor to be considered in otolaryngology–head
and neck surgery units, because inpatients with malignancy and tracheal fenestration,
who lack normal host defense mechanisms in the upper respiratory tracts, are
easily infected with airborne MRSA. Postoperatively, patients with head and
neck cancer should be protected from airborne MRSA infection.
However, there are little extant data about airborne MRSA.5
In this study, therefore, we examined MRSA samples from the air of the rooms
of MRSA-infected or -colonized inpatients of an otolaryngology–head
and neck surgery inpatient unit.
PATIENTS AND METHODS
PATIENTS
Characterization of 3 inpatients who participated in this study is given
in Table 1. The 3 inpatients who
had squamous cell head and neck cancers had been colonized or infected with
MRSA during their stay in the hospital. The number of MRSA isolates in the
clinical samples was more than 106 colony-forming units (CFU) per
specimen; no change in this number was observed during the course of the present
study.
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Table 1. Characteristics of Patients With Methicillin-Resistant Staphylococcus aureus (MRSA) Infection*
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AIR SAMPLING OF BACTERIA
Sampling sites and methods used in this study are summarized in Figure 1. In 37 beds at an otolaryngology–head
and neck surgery unit at a tertiary care medical university hospital, a 6-stage
Andersen air sampler (Nihon Kagaku Kogyo Co, Ltd, Osaka, Japan) was used to
collect air samples at a rate of 28.3 L/min for 30 min/d (total, 849 L)6 from the closed, single-patient rooms of the 3 participating
inpatients. Each of the samples was collected on both Trypto-soya agar (Nissui
Pharmaceutical Co, Ltd, Tokyo, Japan) and MSO agar (salt egg-yolk agar containing
6 mg/L of oxacillin; Nissui Pharmaceutical Co, Ltd). Trypto-soya and MSO agar
plates were used to isolate general bacteria and MRSA, respectively. The sampler
was placed on a rack 1 m above the floor at a distance of 1 m from the bed.
Air sampling was carried out weekly for a month (for a total of 3 sampling
times) in the morning, when the patients were at rest and when the bedsheets
were changed. After the samples had been collected, the culture media were
incubated at 37°C for 48 hours. After incubation, the colonies on the
agar plates were counted and the results were expressed as CFU per cubic meter
(CFU/m3) of air. Gram staining of the isolates on Trypto-soya agar
was performed. The species of the isolates were identified by the methods
described below.
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Figure 1. Sampling sites and methods for
collection of bacteria.
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SAMPLING OF INANIMATE ENVIRONMENTS
Samples were collected from 3 sites (bedsheets, floors, and sinks) in
areas of 10 cm2 in the rooms of each of the 3 inpatients (Figure 1). Trypto-soya and MSO agar plates
were used and the stamp method was chosen to perform sampling. In this method,
agar stamps are rotated several times on the surfaces of the environments.
Sampling was carried out 3 times in duplicate at the same time that the air
was sampled from the rooms. Agar plates were incubated at 37°C for 48
hours. After the incubation, the number of CFU were counted.
ISOLATION OF BACTERIA FROM THE HANDS AND NASAL CAVITIES OF THE PATIENTS
At the same time, air samples and samples from the nasal cavities of
the patients were taken; the latter sampling was performed with sterile cotton
swabs moistened with sterile phosphate-buffered saline.7
The swabs were inoculated on MSO agar and incubated at 37°C for 48 hours.
Samples were taken from the hands of the patients via the stamp method with
MSO agar plates.
IDENTIFICATION OF MRSA
The MRSA isolates were identified 48 hours after the start of incubation
at 37°C on MSO agar plates. The mecA gene was
detected by polymerase chain reaction and primers as previously described.8 Fifty nanograms of bacterial DNA was used as the template
DNA. DNA amplification was carried out for 40 cycles in 50 µL of reaction
mixture as follows: denaturation at 94°C for 30 seconds, annealing at
55°C for 30 seconds, and extension at 72°C for 1 minute, with a final
extension at 72°C for 5 minutes. Ten microliters of the polymerase chain
reaction products was analyzed by 2% agarose gel electrophoresis. The presence
of a 533–base pair amplimer was taken as an indication of the presence
of the mecA gene.
PHENOTYPING OF MRSA
Antibiotic susceptibility was determined by the microdilution broth
method, in accordance with the National Committee for Clinical Laboratory
Standards guidelines.9 The antibiotics used
in the test were ampicillin, piperacillin sodium, oxacillin sodium, cefaclor,
imipenem, cefazolin sodium, flomoxef, cefotiam hydrochloride, gentamicin sulfate,
arbekacin, minocycline hydrochloride, ofloxacin, erythromycin lactobionate,
clindamycin phosphate, vancomycin hydrochloride, and fosfomycin. Coagulase
types were also determined by using coagulase antiserum (Denka Seiken, Co,
Ltd, Tokyo).10
GENOTYPING OF MRSA
Genomic DNA analysis11 was done with
pulsed-field gel electrophoresis (PFGE). Pulsed-field gel electrophoresis
was performed by the procedure described by Struelens et al,12
with some modifications. Cells were treated using Gene Path Group 1 Reagent
Kits (Nippon BIO-RAD Laboratories, Tokyo) and digested with SmaI (Takara Shuzo Ltd, Shiga, Japan). Electrophoresis was performed
on a 1% agarose gel (Nippon BIO-RAD Laboratories) in a CHEF MAPPER system
(Nippon BIO-RAD Laboratories) at 4°C for 22 hours in 0.5x Tris-borate-EDTA
buffer at 170 V; initial and final pulse times were 5 and 80 seconds, respectively.
The gels were stained with ethidium bromide, visualized in a transilluminator,
and photographed with Polaroid film (type 665) in a Polaroid Land camera (Nippon
Polaroid, Tokyo).
RESULTS
SAMPLING OF AIRBORNE BACTERIA DURING THE RESTING PERIOD
Bioaerosol particles were separately collected in a 6-stage Andersen
air sampler according to their aerodynamic diameters (from >7 to 0.65 µm)
(Table 2). The mean ± SD
total CFU/m3 of air on Trypto-soya agar were 130.4 ± 20.2,
117.0 ± 10.3, and 95.8 ± 10.6, respectively, in each of the
single rooms of each of the 3 MRSA-infected patients during the resting period
(Table 2). The main particles
were collected between stage 4 and stage 5 of the air sampler. From 48.1%
to 76.1% of the bioaerosol particles were within a respirable range of less
than 4 µm (stages 4, 5, and 6 in Table 3). Methicillin-resistant S aureus
was detected in a few CFU/m3 of the air samples during the resting
period (Table 2).
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Table 2. Air Contamination With General Bacteria and Methicillin-Resistant Staphylococcus aureus (MRSA) in the Single Rooms of Inpatients
With MRSA Infections During the Period of Rest*
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Table 3. Percentage of General Bacteria-Carrying Particles During the
Rest Period and Bedsheet Changing, by Aerodynamic Size*
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SAMPLING OF AIRBORNE BACTERIA WHEN BEDSHEETS WERE CHANGED
Next, we collected bioaerosols from the single rooms of each of the
3 MRSA-infected patients when the bedsheets were changed; samples were collected
with an Andersen air sampler. As shown in Table 2, the mean ± SD total CFU/m3 of air on
MSO agar was 62.4 ± 8.4, 73.4 ± 14.1, and 58.5 ± 6.2.
During this test period, there were approximately 50 times the number of CFU
as during the resting period. Methicillin-resistant S aureus was isolated in each stage on MSO agar, and the aerodynamic diameters
of the MRSA isolates were mainly more than 5 µm (Table 3). About 20% of the MRSA particles were within a respirable
range of less than 4 µm (Table 3).
SAMPLING OF BACTERIA ON INANIMATE OBJECTS
As shown in Table 4, the
mean ± SD total CFU/10 cm2 on the bedsheets from each of
the 3 patients' rooms, as observed on MSO agar, were 3.0 ± 0.6, 3.2
± 1.1, and 3.3 ± 3.1. Methicillin-resistant S aureus was also isolated from the floors and the sinks. The CFU of
all the environmental samples incubated on Trypto-soya agars exceeded the
upper detection limit of the 1 x 102 CFU/plate (data not
shown).
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Table 4. Methicillin-Resistant Staphylococcus aureus (MRSA) Environmental Contamination in the Single Rooms of Inpatients
With MRSA Infection*
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SAMPLING OF MRSA ON HANDS AND IN THE NASAL CAVITIES OF INPATIENTS
Methicillin-resistant S aureus was detected
on the hands and in the nasal cavities of all of the patients. The mean ±
SD total CFU/10 cm2 on all 3 of the patients' hands, as observed
on MSO agar, were 4.0 ± 3.1, 3.7 ± 2.4, and 8.3 ± 4.1
(Table 4).
ANTIBIOTIC SUSCEPTIBILITY PATTERNS OF MRSA
One clinical isolate from patient 1, 5 of all isolates from the air,
5 of all isolates from the inanimate environments (floor, bedsheets, and sink)
in the room of patient 1, and 1 of all of the isolates from the hands and
nasal cavity of patient 1 were tested for antibiotic susceptibility. As shown
in Table 5, the environmental
isolates had the same susceptibility patterns as the isolates taken from the
room of patient 1. Furthermore, the antibiotic susceptibilities of the 3 clinical
isolates from each patient had the same pattern (data not shown). Most of
the trial antibiotics were not effective against MRSA. Infection or colonization
with MRSA was often resistant to minocycline treatment; however, all MRSA
infections were sensitive to arbekacin and vancomycin therapy. All isolates
had type II coagulases.
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Table 5. Antibiotic Susceptibility of the Methicillin-Resistant Staphylococcus aureus Isolates*
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GENOMIC DNA ANALYSIS OF THE MRSA BY PFGE
As shown in Figure 2, the
environmental isolates, the isolates from the inpatient, and the 3 clinical
isolates exhibited identical restriction fragment patterns after PFGE of SmaI-digested genomic DNA. This result was consistent with
findings from the antibiotic susceptibility assay and coagulase typing.
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Figure 2. Pulsed-field gel electrophoresis
of SmaI-digested genomic DNA from methicillin-resistant Staphylococcus aureus isolates. M indicates  marker; lane 1,
methicillin-resistant S aureus TS0001, clinical isolate
of patient 1; lanes 2-6, TS0002-6, isolates from the air; lanes 7-11, TS0007-11,
isolates from the inanimate environments; lane 12, TS00012, isolate from patient's
hand; lane 13, TS00013, isolate from patient's nasal cavity; lane 14, clinical
isolate of patient 2; and lane 15, clinical isolate of patient 3.
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COMMENT
In this study, we demonstrated that MRSA was recirculated among the
patients, the air, and the inamimate environments, especially when there was
movement in the rooms. This suggests that airborne MRSA may play a role in
MRSA colonization in the nasal cavity or in respiratory MRSA infections.
A standard 6-stage Andersen cascade sampler13
was used for collecting MRSA isolates, which were separated according to aerodynamic
dimensions from the air in the rooms of MRSA-infected or colonized inpatients.
The sampler is widely used in aerobiological studies.6, 13, 14
As a rule, 6-stage cascade Andersen samplers allow for precise microbiological
evaluation as well as for the separation of the organisms according to particle
size. Larger microorganism-carrying particles, in the range of 4 to 8 µm,
are separated in the first 3 stages, whereas smaller particles (<4 µm)
are separated in the 3 lower stages. This allows for the detection and differentiation
of respirable (stages 4-6) and nonrespirable (stages 1-3) particle-adsorbed
microorganisms present in the ambient air. The particles of stages 5 and 6
reach the alveoli. Particles smaller than 5 µm may exist in the air
for a long time and can reach the lungs and cause infection in susceptible
tissues if inhaled.14 As shown in Figure 1 and Table 3, MRSA was detected both during the rest period and when
bedsheets were being changed; the latter period was particularly of interest,
as MRSA of all stages was observed in that period. This finding suggests that
MRSA is able to colonize in the nasal cavity or even reach the lungs. Thus,
it is crucial to design an efficient control system to limit the accumulation
of bacterial cells in environments in which recirculation of air is performed.
The number of CFU of MRSA from air sampling during the changing of bedsheets
in MRSA-infected patient rooms was higher than that observed during the resting
period. When medical staff were present in the rooms of patients, the number
of CFU of MRSA increased in and around the rooms, indicating that MRSA on
surface environments spreads during periods of movement, such as when bedsheets
are changed in hospitals. In such cases, there is also the potential danger
of medical staff acquiring the epidemic strain from a patient by direct contact
and then further risk of transmitting it to other patients.4
Moreover, MRSA may be transferred from one patient to another by airborne
transmission and by direct hand-to-hand contact. Therefore, to prevent the
spread of MRSA, it is recommended that gloves be worn routinely by all personnel
entering the rooms of patients with MRSA.15
More careful disinfection of inaminate hospital environments is also required
for the prevention of airborne transmission of MRSA. Such disinfection procedures
might promote a decline in the nosocomial MRSA infection rate.
Antibiotic effectiveness against MRSA infection was low. However, a
few antibiotics, (minocycline, arbekacin, and vancomycin) were still effective.
All MRSA isolates identified in the ward had one origin, as determined by
the antibiotic pattern. Occurrence of MRSA in the ward was effected by spreading
of a clone. These results indicate that MRSA isolates from the patients, the
air, and the inanimate objects might share a common origin. The classification
of genomic DNA fingerprints by PFGE is proposed as a useful and effective
means for the purpose of epidemiological studies of nosocomial infection of
MRSA.12, 16, 17 Therefore,
we confirmed by PFGE that isolates from the patients, the air, and the inanimate
environments had a common origin (Figure 2). The present findings suggest that MRSA was recirculated among
the patients, the air, and the inanimate objects in the rooms; transmission
was especially likely when there was movement in the rooms.
In this study, we confirmed that MRSA could be acquired by medical staff
and patients through airborne transmission. The findings suggest the importance
of protecting patients against cross-infectious agents existing in aerosols.
Although measures for prevention and control of nosocomial infection with
MRSA include handwashing with an antimicrobial agent; wearing a gown, gloves,
and a mask; and removing MRSA from the nasal vestibule,18, 19
few measures have been established to control airborne bacteria. Laminar unidirectional
airflow, air ventilation, and air filtration could also be beneficial in hospital
environments and should be considered. Further studies will be needed to assess
the levels of MRSA contamination of air and to develop more effective means
of controlling and removing airborne MRSA.
AUTHOR INFORMATION
Accepted for publication November 14, 2000.
The technical assistance of Teturo Muratani in performing PFGE is gratefully
acknowledged.
From the Departments of Otorhinolaryngology (Drs Shiomori and Makishima)
and Microbiology (Dr Miyamoto), University of Occupational and Environmental
Health, School of Medicine, Kitakyushu, Japan.
Corresponding author and reprints: Teruo Shiomori, MD, PhD, Department
of Otorhinolaryngology, University of Occupational and Environmental Health,
School of Medicine, Kitakyushu 807-8555, Japan.
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