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81（ 31 ）〈Brief Report〉

Microfloral analysis of pediatric respiratory syncytial
virus infection, with and without the use of antibiotics
Koo Nagasawa1,2, Kenichi Takeshita3, Hatsumi Taniguchi4,
Hiroshi Suzuki3, Sachiko Naito1, Haruka Hishiki1,
Tadashi Hoshino5, Hirokazu Kimura2,6, Kazuo Suzuki7,
Naoki Shimojo1 and Naruhiko Ishiwada8
1

Department of Pediatrics, Graduate School of Medicine, Chiba University
Infectious Disease Surveillance Center, National Institute of Infectious Diseases
3
Department of Pediatrics, Chiba Rosai Hospital
4
Department of Microbiology, University of Occupational and Environmental Health5
Division of Infectious Diseases, Chiba Childrenʼs Hospital
6
School of Medical Technology, Faculty of Health Science, Gunma Paz University
7
Asia International Institute of Infectious Diseases Control, Teikyo University
8
Department of Infectious Diseases, Medical Mycology Research Center, Chiba University
2(Received for publication October 29, 2017)

Objectives To describe, in detail, the inﬂuence of antibiotics on the microﬂora ofchildren with respiratory syncytial virus (RSV) infection.
Methods Eight hospitalized infants with RSV infection were included in the
study. To examine the change in the microﬂora before and after the use ofantibiotics, we simultaneously collected nasopharyngeal and stool swabs from eachpatient at 3 points; before antibiotics use, during antibiotics use, after use. The use ofantibiotics was determined by clinicians. In 6 patients, antibiotics were used, and in
2, they were not. We analyzed the nasopharyngeal and fecal microﬂora through theclone library method, using ampliﬁed fragments of the 16S ribosomal RNA genewith universal primers.
Results With regards to nasopharyngeal microﬂora, of the 6 patients who wereprescribed antibiotics, 4 had pathogenic bacteria including Haemophilus inﬂuenzaeand Moraxella catarrhalis, on admission, and only 1 patient had M. catarrhalis 3–5
days after admission. However, in 3 patients, M. catarrhalis was the dominantbacterium, 1–2 weeks after hospital discharge. Among the patients who were not*Corresponding author: Naruhiko Ishiwada, Department of Infectious Diseases, Medical Mycology Research Center, Chiba

University, 1–8–1 Inohana, Chuo-ku, Chiba-shi, Chiba 260–8677, Japan. Phone: ＋81–43–226–2799; Fax: ＋81–43–226–2786;
E-mail: ishiwada@faculty.chiba-u.jp

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prescribed antibiotics, the representative pathogens of childhood pneumonia were notobserved in any of the patients 1–2 weeks after hospital discharge, despite them having
those pathogens on admission or 3–5 days after. With regards to fecal microﬂora, thenumber of bacterial species decreased after antibiotics use, except in 1 patient. One
to 2 weeks after hospital discharge, the number of bacterial species was lower thanthat observed on admission, in all the cases in which antibiotics were used.
Conclusions The number of bacterial species both in the nasopharyngeal andfecal samples decreased after antibiotic use. The change in the dominant bacteria of
the nasopharynx may be different between the cases in which antibiotics wereadministered and those in which they were not. These results suggest that clinicians
should administer antibiotics, taking into consideration their inﬂuence on patientsʼ
microﬂora.

1. Introduction
Respiratory tract infections (RTIs) occur commonly in children. RTIs are induced by many
microorganisms, and, in children, viruses are the major cause1). The respiratory syncytial virus(RSV) is one of the major causative viruses of RTI in children1). Generally, patients with RSV infection receive symptomatic treatment. If the patients are considered as having a bacterial co-infection, they are treated with antibiotics. However, it is not always easy to assess if patients havea bacterial co-infection, at the ﬁrst medical examination. Moreover, the bacterial co-infection
rates of RTIs, caused by RSV, vary, by study design1,2). Therefore, in the clinical setting, antibiotics are sometimes prescribed to children with RSV infection, even when they are not required.
The use of antibiotics has its own demerits. Diarrhea is one of the common side effects ofantibiotics use. Although several previously conducted studies have suggested that the use of antibiotics inﬂuences microﬂora, those reports used bacterial cultures3–5). The technique involved inbacterial culture is relatively easy and can be conducted easily in hospitals. However, in bacterial
cultures, non-culturable bacteria cannot be detected, and the percentage of each bacteria in a microﬂora cannot be estimated. To overcome these drawbacks, a method using a sequencer andpolymerase chain reaction (PCR) for 16S ribosomal RNA was developed6). To examine, in detail,the inﬂuence of antibiotics on the microﬂora of children with RSV infection, we analyzed nasopharyngeal and fecal microﬂora simultaneously through this method, before and after antibiotics
were used. In this study, we focused on the relationships between viral infection, use of antibiotics and microﬂora. To the best of our knowledge, there are no reports, till date, on how antibiotics
change both nasopharyngeal and stool microﬂora, using this new molecular biological method.
We aimed to estimate the change in the microﬂora of children with viral infection, and, thereby,
guide the proper use of antibiotics in these patients.

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83（ 33 ）2. Materials and Methods
2.1. Participants
The patients in the present study were hospitalized infants with RSV infection, as diagnosed
by rapid test kits (Chiba University: RSV examen™, Becton Dickinson and Company; Franklin
Lakes, NJ. Chiba Rosai hospital and Chiba Childrenʼs hospital: QuickNavi™ RSV, DENKA
SEIKEN Co., Ltd.: Tokyo, Japan), between September 2014 and March 2015, in Chiba University Hospital, Chiba Rosai Hospital and Chiba Childrenʼs Hospital. We excluded patients inwhom antibiotics were administered within 2 weeks from the date of hospital admission. We also
excluded patients in whom the injection of palivizumab, an anti-RSV monoclonal antibody, wasrecommended. Written informed consent was obtained from the participantsʼ guardians.
2.2. Study protocol
To examine the change in the microﬂora, before and after the use of antibiotics, we collected
nasopharyngeal and stool swabs, simultaneously, from each patient at 3 points; on admission (before antibiotic use), 3–5 days after admission (during antibiotic use), and 1–2 weeks after discharge from the hospital (after antibiotic use). The use of antibiotics was determined by clinicians. The severity of the condition, in each patient, was scored, as previously described7,8). The
study protocols were approved by the Ethics Committee of the Chiba University (approval no.
1857), the Chiba Rosai hospital (approval no. 26–22), and the Chiba Childrenʼs hospital (approval no. 2014-01-02). This study was carried out in accordance with the protocol.
2.3. Analysis of the nasopharyngeal and fecal microﬂora
We analyzed the nasopharyngeal and fecal microﬂora through the clone library method,using ampliﬁed fragments of the 16S ribosomal RNA gene with universal primers9–12). After bacterial cell staining with ethidium bromide, we counted the total number of bacterial cells in thesample13). DNA was extracted from the samples by adding 2 ml of sterilized water and vigorously
shaking the samples with sodium dodecyl sulfate (ﬁnal concentration: 3.0%) and glass beads9,11).
Then, the total number of bacterial cells in the sample was counted again, and we evaluated the
efﬁcacy of cell lysis, as previously described9–12). PCR of 16S rRNA gene was subsequently performed, using universal primers, under the following conditions; 95°C for 5 minutes, 30 cycles at
94°C for 1 minute, 50°C for 1 minute, and 72°C for 2 minutes, followed by a ﬁnal extension at
72°C for 7 minutes11,13). The PCR amplicons were cloned using a TOPO TA cloning kit (Invitrogen; Carlsbad, CA). Of them, a total of 96 colonies were randomly selected for the sequencing
analysis. The analysis was performed using a 3130xl Genetic Analyzer (Applied Biosystems), according to the instrument manual. A Blast search was used to determine the species of each clone
library. These analyses were performed as soon as possible, after the samples were collected.

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3. Results
3.1. Patientsʼ characteristics
A total of 8 patients were assessed in this study; of them, antibiotics were used in 6 (ampicillin; 1 patient, ampicillin/sulbactam; 5 patients). As shown in Table 1, the age of the patients inwhom antibiotics were used was 8.3±5.2 months (mean±standard deviation, SD), while that ofthe patients in whom antibiotics were not used was 4.0 months. The SD of the latter could not becalculated because of the small number. All the patients had a lower respiratory infection, such asbronchitis, bronchiolitis, and pneumonia. With regards to the severity of the RTIs, in this study, 6
out of the 8 patients were categorized as having a mild form of the infection, while 1 each wereconsidered as having moderate and severe infection, respectively.
3.2. Total bacterial cell numbers and cell lysis
All the samples collected in the present study had the sufﬁcient number of bacterial cells foranalysis. The medians of the total bacterial cell number are shown in Table 2. A larger number ofbacterial cells was found in the stool than the nasopharynx. Furthermore, the total number of bacterial cells tended to decrease after admission, regardless of the use of antibiotics. The efﬁciency
of cell lysis ranged from 74.5% to 99.9% (median 91.0%).

Table 1. Clinical features of the patients in the present study

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85（ 35 ）Table 2. The median of total bacterial cell numbers

Fig. 1. The number of nasopharyngeal bacterial species in a) the patients in whom antibiotics
were used and b) patients in whom antibiotics were not used

3.3. Nasopharyngeal microﬂora
We analyzed 22 nasopharyngeal swabs, including 8 samples on admission, 8 samples 3–5
days after admission, and 6 samples 1–2 weeks after hospital discharge. In the case of 2 patients,
samples could not be collected at the time of hospital discharge, as 1 patient was transferred toanother hospital and the other did not allow the collection of nasopharyngeal samples 1–2 weeks
after discharge.
The number of nasopharyngeal bacterial species is shown in Fig. 1. As shown in Fig. 1, thenumber tended to decrease after the use of antibiotics, while the number of patients in whom antibiotics were not used was either constant or tended to increase. Furthermore, we examined thechange in the dominant bacteria, in each sample (Table 3). Of the 6 patients in whom antibiotics
were prescribed, the representative pathogens of childhood pneumonia, including Haemophilus
inﬂuenzae and Moraxella catarrhalis, were noted in 4 patients, on admission, while M. catarrhalis was observed in 1 patient, 3–5 days after admission. However, in 3 patients, M. catarrhalis
was the dominant bacterium 1–2 weeks after hospital discharge. However, among patients who
were not prescribed antibiotics, none of the patients had the representative pathogens of child-Table 3. Dominant bacterium of each nasopharyngeal sample

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87（ 37 ）hood pneumonia 1–2 weeks after hospital discharge, although they had those pathogens on admission or 3–5 days after admission.
3.4. Fecal microﬂora
Twenty-three stool samples, including 8 samples collected on admission, 8 samples 3–5 days
after admission, and 7 samples 1–2 weeks after hospital discharge were analyzed. One sample
could not be obtained due to hospital transfer. The number of bacterial species in each stool sample is shown in Fig. 2. The number decreased after the use of antibiotics, except in the case of one
patient (case 3). However, the number decreased in case 3 too, after hospital discharge. One to 2
weeks after hospital discharge, the number of bacterial cells was lower than that observed on admission, in all the cases in which antibiotics were used. We could not ﬁnd a tendency for the reduction in the number of bacterial cells and species in the no antibiotics admission cases. Furthermore, in the present study, we could not detect the dominant bacterium in the stool.
3.5. Correlations between nasopharyngeal and fecal microﬂora during the clinicalcourseTable 4 shows the number of bacterial species in the nasopharyngeal and fecal microﬂora, in
the patients, during the clinical course. The number of bacterial species in both the nasopharynx
and fecal microﬂora decreased in 4 of 6 patients treated with antibiotics. One to 2 weeks after antibiotic treatment, in 2 specimens—the nasopharynx specimen of case 4 and fecal specimen ofcase 6—were recovered number of bacterial species before antibiotic treatment.
Fig. 2. The number of fecal bacterial species in a) the patients in whom antibiotics were used
and b) those in whom antibiotics were not used

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89（ 39 ）4. Discussion
In this study, we described the change in the microﬂora, in children with a viral infection.
Our main ﬁndings are as follows: First, the number of bacterial species in both the nasopharyngeal and fecal samples decreased after the use of antibiotics. Second, the change in the dominantbacteria of the nasopharynx may be different between cases in which antibiotics were administered and those in which they were not. Reports suggest that the divergence of the microﬂora in
the stool decreased through antibiotic administration14,15). Furthermore, Brook et al and Konno et
al showed, through bacterial cultures, that antibiotics affected the nasopharyngeal microﬂora4,5).
Those studies, however, did not examine the microﬂora of the upper respiratory tract, using molecular biological methods. In contrast, we examined both nasopharyngeal and fecal microﬂora,
simultaneously, using the clone library method. As a result, we could accurately evaluate ﬂuctuations in the microﬂora, in the nasopharynx and stool, including bacteria that are hard to culture.
In the present study, we focused on patients with a viral infection. Hamano-Hasegawa et al.
reported that 15.2% of pediatric patients with community-acquired pneumonia have bacterial coinfection, using the broad-range PCR method1). Hishiki et al. showed that 45% of patients withlower respiratory tract infection, caused by RSV—which is one of the most common causes ofinfection in children—have bacterial co-infection, through the washed sputum culture method2).
In our study too, 5 of 8 patients had the pathogenic bacteria of pediatric pneumonia on admission,and another patient had Streptococcus pneumoniae 3–5 days after admission. However, in the patients in whom antibiotics were not prescribed, those pathogenic bacteria were not detected 1–2
weeks after hospital discharge. Based on this result, it is suggested that not all detected pathogenic bacteria may be causative pathogens in patients with a viral infection; in fact, bacterial infections may be more over-diagnosed than expected.
In this study, the number of bacterial species decreased after the use of antibiotics, in the nasopharyngeal swab samples and stool samples, while the number of bacterial species did not decrease, in cases in which antibiotics were not administered. Many studies reported that the divergence of fecal microbiota decreased through the use of antibiotics14–17). The divergence ofnasopharynx microbiota may also decrease through antibiotics use, with regular dosage.
The dominant bacterium of the nasopharynx differed, depending on the phase at which eachsample was collected. Three to 5 days after admission, in the cases in which antibiotics were administered, the changes in the number of bacterial species could be attributed to the antibacterial
activity of the antibiotics. After the use of antibiotics, pathogenic bacteria, including H. inﬂuenzaeand M. catarrhalis, disappeared. However, M. catarrhalis was recovered 1–2 weeks after hospitaldischarge, in cases in which antibiotics were used. Brook et al suggested that potential pathogens
including S. pneumoniae, H. inﬂuenzae, and M. catarrhalis rebound faster in those treated withamoxicillin/clavulanate compared to those in whom cefdinir was administered, although the90（ 40 ）THE JAPANESE JOURNAL OF ANTIBIOTICS

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study used the bacterial culture method5). We used the clone library method, and, therefore, wereable to evaluate a large number of bacteria, including those species that are difﬁcult to culture.
Our results were compatible with those of the study by Brook et al.5).
To estimate the pathogens in patients with RTI, at the initial diagnosis, may be difﬁcult as
the symptoms of RTI do not vary from those of other microorganisms. In addition, the results ofbacterial culture, which is the standard for bacterial infection, are obtained only after a few days.
A substantial number of instances of bacterial and viral co-infections have been reported1,2).
Therefore, it can be assumed that antibiotics are used in several cases even when they are not required. Some reports suggest that the use of antibiotics in infants may raise the risk ofasthma18,19). It was thought that these demerits were caused by the change in the microﬂora18).
Our results showed that antibiotics may change the microﬂora of both the nasopharynx andbowel.
Our study had some limitations. First, the number of patients in the present study wasslightly small. Second, samples from two patients that could not be collected at all 3 points werealso included in the present study. To conﬁrm these effects of antibiotics on microﬂora, further
studies are needed. Based on the results of the present study, clinicians are urged to use antibiotics taking into consideration their inﬂuence on microﬂora.
Acknowledgements
This work was supported by JSPS KAKENHI Grant Number G17K16288, and Special Research fund of Nurture of Creative Research Leaders in Immune System Regulation and Innovative Therapeutics Leading Graduate School at Chiba University. We would like to thank Editage(www.editage.jp) for English language editing.
Competing interests
The authors declare that they have no competing interests.

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