In the first two years of the study (FY2018 and FY2019), next-generation sequencers were used to establish a
comprehensive technique for sequencing ARGs (metagenomic analysis) in environmental water (Pathogen
Genomics Center, National Institute of Infectious Diseases). Metagenomic analysis was then carried out on
samples of wastewater from sewage treatment plants provided by 34 local governments (219 samples in total,
collected in summer (August) and winter (February) from August 2018 to February 2020). The number of decoded
reads of the ARGs in question was detected based on a database of ARG sequences associated with antimicrobials
used in clinical settings and food-producing animals. In addition, the Reads Per Kilobase of gene per Million
mapped reads (RPKM) method, which normalizes based on ARG base length and total decoded reads in the
metagenome, was used to calculate the relative concentration of ARGs and conduct a comparative analysis
between samples. Last year’s report stated that the ARG levels tended to be slightly higher in winter than in
summer, but ongoing surveillance for two years (a total of four occasions) revealed a continued upward trend in
ARG levels. The primary cause was the detection of significantly high levels (p<0.01) of genes resistant to
sulfonamides and quaternary ammonium salts (disinfectant), with the basic component genes of Class 1 integrons
(sul1, qacEdelta) known to have been widely acquired and propagated among Enterobacteriaceae thought to be a
contributory factor in their increased detection. On the other hand, there has been a marked decline in macrolide
resistance genes in sewage treatment plants and wastewater, which is a result thought to reflect a fall in the use of
macrolides in humans. A similar downward trend was also seen in quinolone resistance genes, suggesting a
relationship to a decline in the use of quinolones in humans. However, a deviation was seen from the situation in
regard to the isolation of quinolone-resistant Escherichia coli. As the research group’s metagenomic analysis
technique focuses on detecting the externally acquired oqx and qnr genes, it did not evaluate mutations in the
quinolone resistance-determining regions (QRDR) of the gyrA and parC genes that are the inhibitory targets of
quinolones. While the frequency of external acquisition has at least declined and might be approaching a desirable
situation, further ongoing surveillance is essential. As the research group’s metagenomic analysis technique
conforms to metagenomic analysis techniques used globally, the study is believed to have provided information
that will be important when comparing reports from different countries. The group plans to continue conducting
nationwide surveillance twice a year (in summer and winter) with the assistance of local governments and put in
place Japanese environmental AMR (resistome) infrastructure.
In terms of global surveillance, Denmark (The National Food Institute, DTU (WHO Collaborating Centre and
European Union Reference Laboratory for Antimicrobial Resistance in Foodborne Pathogens)) is leading a WHOsupported environmental surveillance initiative called the Global Sewage Surveillance Project (GSSP).[1] As this
project targets not only environmental AMR, but also contamination with viruses such as the poliovirus, it is
focusing primarily on inlet water from sewage treatment plants. The first output from the project provided the
results of metagenomic analysis of 79 samples of inlet water from sewage treatment plants (in 60 countries)
collected in January and February 2016.[2] The highest level of ARG contamination among these 60 countries
was 4616.9 FPKM (fragments per kilobase of exon per million reads mapped) in Brazil and African countries also
recorded a high level of ARG contamination, with an average of 2034.3 FPKM. Oceania (New Zealand and
Australia) had the lowest level, with an average of 529.5 FPKM. While Asia (excluding Japan) did not have as
high a level of ARG contamination as Africa, the ARG composition (resistome) was very similar (27%
dissimilarity). ARG FPKM and resistome analysis brought to light results demonstrating a strong correlation
between a country’s population and economic activity on the one hand and its public health measures on the other.
Japan has been involved in this project since 2017, providing pre-treatment inlet water, and a follow-up GSSP
report that includes the evaluation of the Japanese samples is awaited. As the GSSP focuses on (untreated) inlet
water samples from sewage treatment plants, it is difficult to carry out a comparative analysis based on the same
standards as the aforementioned Japanese environmental AMR study, but it does at least provide important
quantitative values for determining whether or not the wastewater from Japanese sewage treatment plants, which
records levels of up to 100 FPKM, necessitates further environmental purification.
In addition to ARG in wastewater, it is vital to identify the presence of ARB that could potentially exist and
proliferate in the environment. Information on the situation within Japan is starting to emerge from the findings of
the aforementioned MHLW research group, including reports that, at a water reclamation center in Tokyo Bay, a
KPC-2-producing Klebsiella pneumoniae strain rarely found in Japan, even in clinical isolates, has been
isolated,[3] that KPC-2 was found in Aeromonas rarely isolated in wound infections,[4] and that E. coli with NDM5 carbapenemase, which has acquired broader-spectrum activity than NDM-1, has been isolated.[5] A report has
also been published on a comprehensive AMR study carried out on hospital wastewater, inlet and outlet water
from sewage treatment plants, and river water in the Yodo River basin in Osaka. Its estimates suggest that a diverse
array of ARB will be isolated from outlet water from sewage treatment plants and that hospital wastewater will
impose an environmental AMR burden unless ozone treatment is carried out.[6] As in the case of the contamination
situation overseas, a more extensive field survey would appear to be required in Japan, at least to ascertain the true
extent of the isolation of ARB in environmental water, and it will be crucial to develop techniques for intensively
eliminating or reducing ARB alone.
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