Prevalence of antimicrobial‐resistant Escherichia coli in endangered Okinawa rail (Gallirallus okinawae) inhabiting areas around a livestock farm

Abstract Antimicrobial resistance (AMR) is an important issue for public, animal and environmental health. It has been suggested that livestock farms could be a source origin of AMR, and some wild animals that inhabit this area may play an important role in the spread of AMR in the natural environment. The prevalence of AMR in Escherichia coli was examined from Okinawa rails (Gallirallus okinawae), an endemic bird in Okinawa Main Island, Japan. Forty‐eight faecal samples of wild Okinawa rails were collected from around a livestock farm area (LA), near human settlements, in which a population of the Okinawa rail had newly inhabited for feeding, and a forest area (FA), their natural habitat. Among 16 E. coli‐positive faecal samples collected around LA, 11/16 (69%) showed antimicrobial resistance and five multiple drug resistance patterns were identified. However, among 15 E. coli‐positive faecal samples from FA, 3/15 (20%) showed antimicrobial resistance, and three multiple drug resistance patterns were identified. These results indicate that the endangered Okinawa rail may also play an important role as a potential vector for the spread of AMR in the natural environment. To maintain ecological health, it is imperative that in situ/ex situ conservation projects that include translocation plans for endangered species are aware of these data.

The Okinawa rail (Gallirallus okinawae) is a flightless bird in the family Rallidae and the species inhabit the evergreen laurel forest zone, called "Yambaru," located in the northern part of Okinawa Main Island, Japan (Figure 1). Wild populations of the Okinawa rail are declining because of habitat loss and predation by invasive alien species such as the small Indian mongoose (Herpestes auropunctatus), feral cats (Felis catus) and feral dogs (Canis lupus) (Arcilla, Choi, Ozaki, & Lepczyk, 2015;Harato & Ozaki, 1993;Yamada & Sugimura, 2004).
The Okinawa rail is currently listed as an endangered species in The International Union for Conservation of Nature Red List (IUCN, 2017) and highly protected under Act on Conservation of Endangered Species of Wild Fauna and Flora and Programs for the Rehabilitation of Natural Habitats and Maintenance of Viable Populations of Japan.
Recently, it has been observed that the Okinawa rail population is slightly increasing around some livestock farms, where they possibly have easy access to food sources, including earthworms and insects (Ogura, Iijima, Ozaki, Nagamine, & Kuwana, 2009). Therefore, it is assumed that the Okinawa rail is exposed to AMR through food and through water contaminated with the faeces of livestock animals.
The aim of this study was to understand whether the Okinawa rail, an endangered species, plays an important role in carrying and spreading AMR, acting as a vehicle between human habitation and the natural environment in Okinawa Main Island. were frequently observed and 29 faecal samples were collected from a forest area (abbreviated FA) in 2014, located about 3-7 km from the LA sites ( Figure 1). It is said that the territory size of Okinawa rails is about 1 km, but it is unpublished (private message from observer).

| MATERIAL S AND ME THODS
Samples were stored at 4°C until laboratory examination.
To identify whether faecal samples were derived from Okinawa rails, we first screened for its characteristic odour in the sampling area. Secondly, molecular methods were employed to confirm that the faecal samples were from the desired species. The whitebreasted waterhen (Amaurornis phoenicurus), in the family Rallidae, inhabits the same area as the Okinawa rail and faeces from this species were used for discrimination. White-breasted waterhen faecal samples were collected from captive individuals at Neo Park Okinawa, Okinawa, Japan, on November 4, 2013. For polymerase chain reaction (PCR) amplification, a primer set targeting a conserved region of the Okinawa rail mitochondrial ATP6 gene was designed using available sequence data (GenBank accession no. AP010821). The primers used were as follows: yanbaru kuina F (5′-ATGGGCCCTAACACTCTCCT-3′; nucleotides 12,945 to 12,964) and yanbaru kuina R (5′-GGAGACTGCGGGTATGATGG-3′; nucleotides 13,311 to 13,292). PCR products were purified using the QIAquick PCR Purification Kit ® (QIAGEN, USA) and direct-sequenced using an ABI 3130xl sequencing system (Applied Biosystems, USA). The described primer set was also used for sequencing.
Okinawa rail faecal samples, identified using PCR, were cultured on MacConkey agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) and screened for E. coli using Triple Sugar Iron and Motility-Indole-Lysine media (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), followed by identification using the ID-Test/EB20 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). The drug susceptibility of Chi-squared testing was used to compare the prevalence of antimicrobial-resistant bacteria from faecal samples obtained from the LA and FA samples. Antimicrobial resistance was expressed as odds ratios with 95% confidence intervals.  Table 1). The prevalence of AMR E. coli in faecal samples from wild Okinawa rails from LA (69%) was significantly higher than those from FA (20%) (p = .006). The MICs of antimicrobials (in μg/ml) are shown in Table 2. Furthermore, five different multiple drug resistance (MDR) patterns were found in LA samples and three different MDR patterns were found in FA samples (Table 3).

| RE SULTS AND D ISCUSS I ON
Previous research investigating AMR in Japanese wild birds revealed that antimicrobial-resistant strains of E. coli were found in 12.5% of wild green pheasants (Phasianus versicolor) and in 15.8% of wild bamboo partridges (Bambusicola thoracicus) (Kanai, Hashimoto, & Mitsuhashi, 1981;Nakamura, Yoshimura, & Koeda, 1982). By comparison, the prevalence of AMR in Okinawa rails from LA was much higher, and it is likely that livestock farms could be a source of AMR exposure. Indeed, it is indicated that resistant strains of bacteria which was produced in the livestock body by inadequate use of antibiotics for the treatment of clinical disease, as feed additives for the prevention of disease, or for nutritional purposes (Blanco, Lemus, & Grande, 2009) and/or which was produced by remained antibiotics in their excrements and feed have mainly emerged from livestock farming to the environment. Our results suggest that Okinawa rails are exposed to AMR E. coli at livestock farms. Therefore, it is suspected that the movement of Okinawa rails between LA and FA transmitted AMR.
The most frequently detected AMR strain of E. coli from wild Okinawa rails was OTC-resistant. OTC is widely used in the rearing of domestic animals to prevent and treat infectious diseases and to promote their growth (Asai, 2010;Horie & Takegami, 2006).

monacha) that inhabit cattle farms in the Kagoshima Prefecture,
Japan. The greatest OTC MIC value in hooded cranes was 128 μg/ml (Kitadai, Obi, Yamashita, Murase, & Takase, 2012); in Okinawa rails, we detected a higher resistance to OTC (MIC ≥ 256 μg/ml). Oonaka, Furuhata, Kiuchi, Hara, and Fukuyama (2004)  Furthermore, CP resistance was detected in rails collected in FA at high rates (67%). CP has been frequently used as a therapeutic TA B L E 1 Proportion of Okinawa Rail fecal samples demonstrating resistance to each antimicrobial. No resistance to CZOP, GM, FOM, SM, CAZ, CTRX, CTX and CXM was detected in Escherichia coli isolates from Okinawa rail faecal samples in both areas  (0) agent for domestic animals in livestock farms in Japan because of its broad-spectrum activity against pathogenic bacteria (Harada, 2009).
However, this drug is toxic to humans, causing aplastic anaemia, and the Japanese government has permanently banned the use of CP for the treatment of disease in food-producing animals (Gilmore, 1986;Harada, 2009 (Sarmah, Meyer, & Boxall, 2006). There were 2-8 multiple drug resistance patterns, including synthetic antimicrobial agents, in resistant E. coli isolated from FA and LA.
It is suggested that AMR occurs as a result of interactions between microbial agents, host organisms and the environment (Delport, Harcourt, Beaumont, Webster, & Power, 2015). The indiscriminate use of certain antimicrobials in human and veterinary medicine has become a significant public health concern because it may select for resistant bacterial strains (Hiltunen, Virta, & Laine, 2017  in different year and our sample size was small. Therefore, more samples need to be collected and genetic analysis of AMR E. coli using the PFGE (Pulsed-Field Gel Electrophoresis) method needs to be performed. Conclusively, it is imperative that attention has to be given to the prevalence of AMR among endangered species in programmes of in situ/ex situ conservation that include reintroduction, in order to maintain public and ecological health.

ACK N OWLED G EM ENTS
The

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests to declare.