J Infect Chemother 22 (2016) 733e737

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Journal of Infection and Chemotherapy

journal homepage: http://www.elsevier.com/locate/jic

Original Article

Effectiveness of weekly polymerase chain reaction-based open

reading frame typing analysis of all newly isolated
methicillin-resistant Staphylococcus aureus strains for controlling
nosocomial infections
Kiyotaka Nakaie a, Koichi Yamada a, b, Keunsik Park c, Yasutaka Nakamura a, d,
Yasuyo Okada a, Akiko Fujita a, Hiroki Fujimoto a, b, Yukihiro Kaneko e, Hiroshi Kakeya a, b, *
a
Department of Infection Control and Prevention, Osaka City University Hospital, Japan
b
Infection Control Science, Graduate School of Medicine, Osaka City University, Japan
c
Department of Medical Quality and Safety Science, Osaka City University, Japan
d
Department of Pharmacy, Osaka City University Hospital, Japan
e
Bacteriology, Graduate School of Medicine, Osaka City University, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Polymerase chain reaction (PCR)-based open reading frame typing (POT) helps differentiate between
Received 2 March 2016 bacterial strains based on the open reading frames (ORFs) of the prophage-encoding genes; multiplex
Received in revised form PCR screening is performed to identify strains based on keeping patterns.
12 July 2016
At our hospital, surveillance of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) trans-
Accepted 13 July 2016
Available online 28 September 2016
mission is undertaken using POT to conduct molecular epidemiological analysis for all newly detected
MRSA strains. In 2014, we performed POT only once a month; however, in 2015, we increased the fre-
quency of POT to once a week, which helped us detect nosocomial transmission that would normally be
Keywords:
POT (PCR-based open reading frame typing)
difficult to detect, and thus achieve 40% reduction in nosocomial transmission, compared to that in 2014.
Methicillin-resistant Staphylococcus aureus This suggests that weekly POT screening for all MRSA strains is one of the effective methods available for
(MRSA) minimizing nosocomial transmission of MRSA.
Nosocomial infection control © 2016 The Author(s). Published by Elsevier Ltd on behalf of Japanese Society of Chemotherapy and
Molecular epidemiological analysis The Japanese Association for Infectious Diseases. This is an open access article under the CC BY-NC-ND
Outbreak license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction transmission of MRSA. The drug susceptibility patterns of MRSA strains

Nosocomial infections caused by various resistant bacteria have
been a source of concern in recent times. Currently, methicillin-
resistant Staphylococcus aureus (MRSA) is the most important
pathogen causing nosocomial infections. To prevent nosocomial
infection, hospitals undertake regular surveillance to detect resis-
tant bacteria, including MRSA.
At our hospital, if MRSA is isolated from newly admitted patients
within 48 h of admission, we define them as “imported cases”; all other
MRSA cases are grouped under “nosocomial transmission.” However,
not all patients undergo MRSA screening upon hospitalization, and
therefore, it is very difficult to accurately assess the nosocomial
* Corresponding author. 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan.
Fax: þ81 6 6646 6056.
E-mail address: kakeya@med.osaka-cu.ac.jp (H. Kakeya).
help us differentiate among isolates. However, some strains might
have identical gene patterns, but they might show different drug
susceptibility patterns; therefore, caution must be exercised when
assessing nosocomial transmission based on drug sensitivity tests
alone. Pulsed-field gel electrophoresis (PFGE) is a highly reliable
method for assessing nosocomial transmission; however, it is time-
consuming and expensive and requires specific skills [1].
The polymerase chain reaction (PCR)-based open reading frame
typing (POT) method, developed by Suzuki et al. [2,3], is based on
phage open reading frame (ORF) typing and can differentiate
among MRSA strains. The ORFs of phage genomes lysogenized in
the MRSAs are amplified in this reaction. POT assay yields results in
only 4 h, and these results are converted into numerical values,
which allows easy comparison of previous data and data analyzed
at other facilities. Furthermore, the discriminatory power of the
POT method is reported to be equal to that of PFGE [1].

http://dx.doi.org/10.1016/j.jiac.2016.07.007
1341-321X/© 2016 The Author(s). Published by Elsevier Ltd on behalf of Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
734 K. Nakaie et al. / J Infect Chemother 22 (2016) 733e737
In the present study, we discuss the use of POT to analyze new
MRSA strains as well as the efficacy of weekly analysis of all strains.
2. Materials and methods
2.1. Reagents and devices
To perform POT, we used a Cica Geneus Staph POT kit (Kanto
Chemical Co., Ltd., Tokyo, Japan) and Cica Geneus DNA Extraction
Reagent (Kanto Chemical Co., Ltd.). PCR was performed using 2720
Thermal Cycler (Thermo Fisher Scientific Co., Ltd., Waltham, MA,
USA). For electrophoresis, Reliant™ Minigel TBE (Lonza Japan Co.,
Ltd., Tokyo, Japan) and Mupid-exU (Mupid Co., Ltd., Tokyo, Japan)
were used. FAS-IV (Nippon Genetics Co., Ltd., Tokyo, Japan) was
used for capturing images. Bacterial identification and drug
susceptibility tests were performed with MicroScan WalkAway-96
SI (Beckman Coulter, Inc., Brea, CA, USA) using MicroScan Series
Panel PC1T (Beckman Coulter, Inc.).
We performed POT according to the manufacturer's instructions
to study MRSA isolates. In brief, the POT kit comprised two multi-
plex PCR reactions, and the output was determined by the presence
of 22 targets. The band size of these products was estimated with
the help of the positive control. The results are expressed as three
POT scores calculated in a binary manner [2].
2.2. Frequency of POT assay to analyze MRSA strains
At our hospital, the results of microbiological tests can be
viewed in in-hospital electronic medical records; however, for
direct confirmation with staff, the Infection Control Team reports
the results to the ward on the day MRSA is isolated, thereby
enabling swift contagion countermeasures.
We began using POT for genetic analysis of strains during
nosocomial MRSA outbreaks in 2010. Since 2014, however, we have
been using POT to analyze all MRSA strains detected. Furthermore,
in 2015, we increased the frequency of POT testing from once a
month to once a week.
If MRSA strains with the same POT number are detected in the
same ward, this information has to reach the medical staff in order
to draw their attention to the spread of MRSA. Moreover, such data
are reviewed weekly by the Infection Control Team, which proac-
tively intervenes if there is a possibility of an outbreak.
2.3. Percentage of MRSA among S. aureus strains
To assess the effectiveness of weekly POT screening for all MRSA
strains, we compared the percentage of MRSA among S. aureus as
observed before regular testing of all strains (2013) to that with
monthly (2014) and weekly testing (2015). The formula used was
(Number of patients testing positive for MRSA/Number of patients
testing positive for S. aureus)  100.
2.4. Conventional definition of MRSA outbreak and nosocomial
transmission
An MRSA outbreak has been defined as three or more cases of
what is considered the same strain of MRSA within four weeks in
one ward. The definition was based on a notice issued by the Health
Policy Bureau, Japan's Ministry of Health, Labour and Welfare in
December 2014. MRSA nosocomial transmission has been defined
as the detection of two or more cases considered to be the same
MRSA strain within six months in our hospital.
2.5. Transmission rate in the same ward
To assess the effects of POT screening frequency, we compared
the transmission rate of MRSA strains in the same ward from
January to October 2014 (monthly screening) and January to
October 2015 (weekly screening). “MRSA transmission in the same
ward” has been defined as two or more cases of which are
considered the same POT strain of MRSA within six months in same
ward.
The transmission rate was calculated using the formula (Num-
ber of cases with the same POT number in the same ward [case 2
onward]/Number of newly admitted patients with MRSA)  100.
Statistical analysis was performed using SPSS ver. 20. The yearly in-
ward transmission rates of MRSA strains were compared using chi-
square test, with P < 0.05 being considered statistically significant.
2.6. Definition of MRSA infection
We defined MRSA infection as follows. Bacteremia was defined
as one or more positive blood cultures from patients with clinical
signs of infection such as fever, chills, and sweats, with or without
local signs and symptoms [4]. Pneumonia was defined as devel-
opment of a new pulmonary infiltrate based on radiographic im-
aging in conjunction with clinical signs, and MRSA was isolated
from a purulent respiratory secretion. Wound infection was defined
as isolation of MRSA from a purulent fluid drained from the wound.
Even if MRSA was isolated from various types of specimens, it was
defined as “colonized” when there were no clinical signs of infec-
tion [5].
3. Results
3.1. Usefulness of POT assay for molecular epidemiological analysis
of nosocomial MRSA strains
Molecular epidemiological analysis of newly isolated MRSA in
2014 and 2015 was performed using POT. The POT types of MRSA
isolates in 2014 and 2015 are listed in Tables 1a and b. The most
frequent isolate detected in 2014 was 106-137-80 (13.5%). Subse-
quently, 93-191-103 (9.1%) and 93-137-103 (5.6%) were isolated.
Similarly, the most frequent isolate detected in 2015 was 106-137-
80 (12.4%). Subsequently, 106-9-80 (4.0%) and 93-217-111 (3.6%)
were isolated. The frequencies of POT types with a unique POT
number were 35.3% and 38.2% in 2014 and 2015, respectively. We
examined the instances of nosocomial transmission of MRSA
strains isolated within 48 h of hospital admission and strains iso-
lated after >48 h of admission. In 2014, MRSA strains with the same
POT number were detected in the same ward in 76 cases, and
therefore, these were suspected to be cases of nosocomial trans-
mission. Of these 76 cases, MRSA was isolated after >48 h of
admission in 57 cases (75%), whereas it was isolated within 48 h in
19 cases (25%); the latter would thus not conventionally be
assessed as nosocomial transmission.
3.2. Reduction in the rate of MRSA among all S. aureus isolates
The rate of MRSA among all S. aureus isolates was 43.8% (271/
619) in 2013, 39.5% (281/712) in 2014, and 33.5% (259/772) in 2015.
The difference between the rate in 2013 and 2014 was not statis-
tically significant; however, the rate in 2015 was significantly lower
than that in 2014 (Fig. 1).
 K. Nakaie et al. / J Infect Chemother 22 (2016) 733e737 735

Table 1a Table 1b
List of POT types in 2014. List of POT types in 2015.

POT number Number of isolates % POT number Number of isolates %

106-137-80 34 13.5 106-137-80 31 12.4

93-191-103 23 9.1 106-9-80 10 4.0
93-137-103 14 5.6 93-217-111 9 3.6
93-185-124 8 3.2 93-137-103 8 3.2
93-217-56 8 3.2 106-153-115 7 2.8
106-9-80 7 2.8 93-153-125 6 2.4
93-189-125 6 2.4 93-191-103 6 2.4
93-153-125 6 2.4 106-9-2 5 2.0
93-217-111 5 2.0 93-253-48 5 2.0
93-189-111 4 1.6 93-155-111 3 1.2
93-191-125 4 1.6 93-137-2 3 1.2
106-153-115 4 1.6 93-223-125 3 1.2
93-149-109 4 1.6 106-137-113 3 1.2
93-223-111 4 1.6 93-217-56 3 1.2
93-137-2 3 1.2 104-9-80 3 1.2
93-217-107 3 1.2 108-8-2 3 1.2
93-153-127 2 0.8 93-219-125 3 1.2
106-9-34 2 0.8 106-153-121 2 0.8
93-181-109 2 0.8 93-187-125 2 0.8
108-8-16 2 0.8 106-77-113 2 0.8
93-147-109 2 0.8 93-221-111 2 0.8
70-18-81 2 0.8 93-189-111 2 0.8
93-153-56 2 0.8 98-155-56 2 0.8
93-245-109 2 0.8 93-200-103 2 0.8
93-183-101 2 0.8 106-137-84 2 0.8
93-183-37 2 0.8 93-183-101 2 0.8
106-153-120 2 0.8 106-29-116 2 0.8
93-253-125 2 0.8 93-187-56 2 0.8
98-187-88 2 0.8 106-59-88 2 0.8
Othersa 89 35.3 93-189-61 2 0.8
Total 252 100.0 106-217-120 2 0.8
a 93-191-125 2 0.8
MRSA isolates with a unique POT number.
93-153-56 2 0.8
106-183-37 2 0.8
3.3. Transmission rates in the same ward 106-73-80 2 0.8
93-157-56 2 0.8
MRSA isolates with the same POT number from the same ward 93-153-120 2 0.8
were isolated, suggesting that nosocomial transmission occurred 106-11-0 2 0.8
93-223-111 2 0.8
within the ward. In 2014, MRSA strains with the same POT number
Othersa 96 38.2
were isolated in the same ward in 28% (70/252) cases, with 22 cases Total 251 100.0
of MRSA infection. In 2015, this percentage was 16% (40/251), with a
MRSA isolates with a unique POT number.
seven cases of MRSA infection. Thus, the rate of detection of MRSA
with the same POT number in the same ward was significantly
lower in 2015 than in 2014 (P < 0.05) (Fig. 2).
(%)
50.0
3.4. Detection of nosocomial transmission by POT screening for all
new MRSA strains
40.0 43.8
39.5
POT screening for all MRSA strains revealed a nosocomial MRSA
30.0 33.5
transmission, which could not have been detected based on the
conventional definition. Two successful cases have been described 20.0
below.
Examples of controlling infections via POT screening for all new 10.0
MRSA strains.
0.0
1) In ward A (emergency ward), MRSA was isolated from 1 to 2 MRSA rate 2013 2014 2015
patients every month; this did not meet the conventional defi-
nition of an outbreak, and therefore, infection control measure Fig. 1. Changes in MRSA isolation rate. MRSA detection rate: (Number of patients
was not reinforced. However, POT screening for all new MRSA testing positive for MRSA/Number of patients testing positive for S. aureus)  100 (%).
strains showed that a single MRSA strain with the same POT
number (106-153-115) was isolated multiple times over a long with the same POT number (106-137-80) were isolated multiple
period (Fig. 3). Infection control measures for MRSA were sub- times, each one case in September and November 2013, in
sequently enforced, and the strain was not isolated again (data February and March 2014, respectively (Fig. 4a). One and three
not shown). MRSA strains with the POT number 106-137-80 were also iso-
2) In Department B (surgical ward), MRSA was isolated from a few lated in ward C (otolaryngology ward) in January to June, and in
patients every month until August 2013, but this did not meet July to December 2013, respectively (Fig. 4b). To determine the
the conventional definition of an outbreak. However, POT transmission route, an environmental investigation was per-
screening for all new MRSA strains revealed that MRSA strains formed in the otolaryngology outpatient ward. MRSA strains
736 K. Nakaie et al. / J Infect Chemother 22 (2016) 733e737

300 30

250 25

200 20

150 15

100 10

50 5

0 0

Number of cases with the same Number of cases with Rate of detection of the same
POT number in the same ward different POT number POT number in the same ward

Fig. 2. Changes in the rate of isolation of MRSA with the same POT number in the same ward. Black column indicates the number of MRSA strains with the same POT number in the
same ward. Gray column indicates the number of MRSA strains with different POT numbers. Detection rate of the same POT number: (Number of cases with the same POT number
in the same ward [Case 2 onward]/Number of newly admitted patients with MRSA)  100 (%).

0 Jul Jan
Aug Sep Oct Nov Dec Feb Mar Apr May Jun Jul Aug
2014 2015
Other strains 3 1 1 3 0 0 2 1 1 1 0 1
106-153-115 strain 1 0 0 1 0 1 0 0 0 1 0 1 0 2

Fig. 3. Number of cases with MRSA in ward A. Black column indicates the number of MRSA strains with the same POT number (106-153-115). Gray column indicates the number of
MRSA strains with other POT numbers. An MRSA strain with the same POT number (106-153-115) was isolated multiple times from July 2014 to August 2015 from ward A.

a 7 b
30
6
25
5
20
4
Number of cases

15
3
10
2

1 5

0 0
Jun Jan Jan-Jun Jul-Dec
201 Jul Aug Sep Oct Nov Dec 201 Feb Mar Apr 2013 2013
3 4 Other strains 13 22
Other strains 1 2 1 4 2 3 1 2 5 1 2 106-137-80
1 3
strain
106-137-80 strain 1 1 1 1

Fig. 4. Number of MRSA cases in ward B (surgical ward) (a) and ward C (otolaryngology ward) (b). Black column indicates the number of MRSA strains with the same POT number
(106-137-80). Gray column indicates the number of MRSA strains with other POT numbers. An MRSA strain with the same POT number (106-137-80) was isolated in ward C in 2013
(b). In ward B, the same strain was also isolated from four patients who visited ward C for pre-surgery examination (a). An MRSA strain with the same POT number (106-137-80) was
also isolated in the environment of ward C.
 K. Nakaie et al. / J Infect Chemother 22 (2016) 733e737
with the same POT number (106-137-80) were isolated in the
environment of the ward C (on the keyboard of the computer,
desk, surface of the scope). Patients carrying MRSA with the POT
number 106-137-80 in Department B had undergone pre-
surgical otolaryngological check during hospitalization. Rein-
forcing infection control in the outpatient Department C
reduced the MRSA isolation rate in both Department B and C
(data not shown).
4. Discussion
The usefulness of POT screening in controlling nosocomial in-
fections has been previously reported. In one of these studies, an
environmental investigation was performed during an MRSA
outbreak; POT screening of MRSA revealed the infection source and
route, resulting in termination of the outbreak [6]. In another study,
POT screening was introduced as a method for identifying trans-
mission within 48 h of hospital admission as nosocomial spread [7].
In addition, POT method considers infection factors such as places
(wards and transfer) and time (date of admission and date of
sample collection), and the number of suspected cases of nosoco-
mial transmission reduced from 118 to 38 [7]. In another study, POT
screening was reported to be useful by enabling swift intervention
via reports based on the objective data on nosocomial transmission
of MRSA [8]. Thus, POT is considered effective for controlling
nosocomial MRSA infections; however, no previous study has
assessed the efficacy of routine POT analysis of all new MRSA
strains. In the present study, POT screening for all newly detected
strains of MRSA revealed the presence of identical strains that were
not detected by conventional culture and susceptibility tests. In
addition, by increasing the frequency of POT screening from once a
month to once a week, we were able to initiate infection control
earlier and reduce in-ward transmission by 40%. Furthermore,
although POT screening for all MRSA strains alone failed to help
achieve a significant reduction over time in the percentage of MRSA
among S. aureus, a statistically significant reduction was observed
after increasing the frequency of POT screening to once a week.
These results suggest that frequent routine POT screening is the key
to control nosocomial transmission of MRSA. Rapid reporting of
POT results to the wards and reinforcing infection control should
help reduce the nosocomial transmission of MRSA. In addition,
increased use of alcohol for hand hygiene in our hospital may have
contributed to the control of nosocomial MRSA infections. The
numbers of hand hygiene opportunities per health worker per
patient-day in our hospital were 6.6 in 2013, 10.1 in 2014, and 9.9 in
2015. The numbers soared between 2013 and 2014; however, the
number in 2015 was almost same as that in 2014. The weekly POT
was started in January 2015. Furthermore, the initiation of weekly
POT reports may have contributed to more careful environmental
cleaning and patient cohorting [9]. This may explain why the same
POT strain decreased, whereas the frequency of handwashing not
increased between 2014 and 2015 (data not shown).
In the present study, POT screening for molecular epidemio-
logical analysis of all new MRSA strains was effective for controlling
nosocomial infections. In a study by Kobayashi et al., the cost of
treatment for one patient with MRSA infection was estimated to be
¥4 million (~$34,000) [10]. As shown in the present study, our
measures reduced the number of cases of nosocomial MRSA
infection from 22 (2014) to 7 (2015), thereby potentially saving a
total of ¥60 million (~$511,000). The cost of the POT kit and reagents
was ¥352400 in 2014 and was ¥483000 in 2015. Taking into
consideration personnel expenses, the use of weekly POT may be a
cost-effective strategy in regions or countries with a high incidence
of the nosocomial spread of MRSA.
737
Although MRSA transmission surveillance by POT screening
could be useful, there are limitations to this approach. Because
MRSA strains with the same POT number 106-137-80 were
frequently isolated, we could not always determine the source and
route of transmission. This can be attributed to insufficient inves-
tigation of the nosocomial transmission as well as failure to identify
the strains rampant in other facilities or in the community. For
more effective infection control of MRSA in the future, it is
important to share available information about the POT numbers of
MRSA among regional hospitals and facilities. These isolates might
act as endemic clones of MRSA. Therefore, we re-analyzed the rate
of detection of MRSA with the same POT number excluding the
most frequent strain (106-137-80) in the same ward. In 2014, the
percentage was 21% (46/218), with 16 cases of MRSA infection, and
in 2015, the percentage was 12% (26/209), with five cases of MRSA
infection. Thus, the rate of detection of MRSA with the same POT
number without 106-137-80 strains in the same ward was signifi-
cantly lower in 2015 than in 2014 (P < 0.01). These data suggest that
the weekly POT may have contributed to the prevention of MRSA
transmission and infection in the nosocomial setting.
We conclude that weekly POT of all new isolates of MRSA may
help to identify the particular MRSA clone provoking nosocomial
transmission in an easier and timelier manner, and that rapid pe-
riodic notification and surveillance are important for effective
control of nosocomial infections.
Conflict of interest
The authors declare that they have no competing interests.
Acknowledgments
This research is supported by the Research Program on
Emerging and Reemerging Infectious Diseases from Japan Agency
for Medical Research and Development (AMED).
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