ISCUSSION

The prevalence of antimicrobial‑resistant E. coli^was very low in all research

sites, ^even though most sampling ^{areas are} open ^torthe public and ^some feces were

collected from culled/captured animals for pest control. This result suggests that the other factors might be more important than the mere "presence" of human activities in the spread of antimicrobial‑resistant bacteria in wild animals under ^some circumstances.

Genetic

diversity

of E. coli ^was observed between different host species

(Fig.

lO),

suggesting that the predominant E. coli strains in intestinal micro flora might be

strains failed to colonize the intestines of adult humans and mice harboring a full intestinal micro flora

(97).

^Therefore, E. coli strains from humans or domestic animals

are unlikely ^to colonize wild animals. However, it was not clear whether the E. coli isolates in the present study ^were resident strains ^or transient strains in intestinal

micro flora. It is needed ^to investigate how long the transient strains ^are sustained in the intestines of animals by continuous sampling from identified animals and the

experimental attempts of E. coli colonization. Comparing

genotypes

of E. coli between individuals of each host species, E. coli isolates from macaques seemed ^to be

species‑specific

(Fig.

^10,

#1‑4),

whereas those from deer, serows and hares were more

specific ^to individuals than ^to species

(Fig.

^{8, #1‑4; Fig. 10,}

i‑1‑2).

^Japanese macaques generally live in groups

(29)

and show social behavior with close ^contacts such ^as

grooming within ^{a same} group. Sika deer also live in groups

(76),

^{but their contacts are}

not ^as close ^as those of macaques. Japanese hares and ^{serows are} usually solitary ^or in small groups composed of mother and offspring

(81, 104).

^Such differences in animal behavior can affect intra‑species transmission ofE. coli. In this study, genetic diversity

within ^an individual was observed in deer, hare, raccoon dog, copper pheasant, bear and macaque

(Figs.

8 and 10,

*).

^This result suggests that undetected antimicrobia1‑resistant strains may exist in E. coli populations of wild animals and be predominant when host animals ^were exposed ^to antimicrobials. Both host factors

(host

species and animal

behavior)

^{and bacterial factors}

(genetic diversity

in intestinal E. coli

population)

^can

affTect the spread of antimicrobial‑resistant bacteria in wild animals.

No tet or bla genes ^were detected in susceptible E. coli, ^even in the strains isolated from the same fecal sample carrying ^an OTC‑resistant strain. Without the

selective pressure of antimicrobials, plasmid‑bone resistance genes such ^as

tet(B)(18)

and blaTEM

(24)

might be rarely transferred ^or maintained in E. coli populations of wild

40

animals.

An OTC‑resistant E. coli ^was isolated from deer in Rausu. Tetracyclines

(TCs)

^{are the most} commonly used antimicrobials in veterinary medicine

(40),

and TC‑resistant bacteria are commonly detected in food amimals in Japan

(40).

^{Rausu has a}

small dairy industry

(S9)

^{and wild deer} occasionally graze in pastures

(S11).

^Therefore,

it is possible that OTC‑resistant E. coli ^were transiently transmitted from cattle ^to wild deer or that the TC residues excreted from cattle in the environment put selective

pressure ^on the E. coli population of deer.

Tet(B)

^{is the most common} determinants of TC‑resistance detected in TC‑resistant E. coli from various host animals

(14).

^{In a} study of the wild small mammals in Canada,

tet(B)

^{was more frequently detected} in isolates obtained ^near swine farms than in isolates obtained from natural ^areas

(57).

^This

supports the hypothesis that the OTC‑resistant isolate from deer in the present study ^was derived from livestock. However, OTC is produced by the soil bacteria, Streptomyces

rimosus

(18).

^Therefore, it is also possible that the OTC‑resistant E. coli from deer was

unrelated ^to the use of TC in veterinary medicine. Further research is needed ^on wild

animals ^near livestock facilities or veterinary clinic, in order ^to determine to what ^extent TC‑use is associated with TC‑resistant E. coli in wild animals.

Four isolates which ^are resistant ^to three antimicrobials

(ABPC,

^NA and

ERFX)

^{were isolated from a raccoon dog} in Shimokita. As far as we know, this is the flrSt report Of fluoroquinolone

(FQ)‑resistant

^E. coli from wild animals in Japan.

FQs

are synthetic antimicrobials. Multidrug‑resistant E. coli isolates in this study ^were highly mutated and they ^are unlikely ^to acquire quinolone‑resistance by only

coincidental point mutations without selective pressure by ERFX or other

FQs.

^The

mutation

type

^observed in this study

(S83L

and D87N in GyrA, and S80I and E84V in

and Japan

(44,95).

^{The different mutation}

type

of four amino acid substitutions

(S83L

and D87N in GyrA, and S80I and E84G in

ParC)

^{was detected in}

FQ‑resistant

^isolates

from human patients

(44,

^95,

100)

and infected dogs

(33).

^Therefore, it is probable that the occurrence of the multidrug‑resistant isolates from a raccoon dog in this study ^were

associated with

FQ‑use

^{in human or} veterinary medicine. Raccoon dogs use a wide range of habitats including farmlands and human settlements, and their home range size varies from 10 to 600 ha

(88).

^Three small villages ^were located within 5 km from the sampling point of the ^raccoon dog's sample carrying resistant strains

(data

^not

shown).

Therefore, it is possible that antimicrobia1‑resistant strains ^were transmitted from humans or domestic animals treated with

FQs

^{to the raccoon dog. ERFX} is mainly eliminated via renal mechanisms

(65).

It is also possible that the environment in the

raccoon dog's home range ^was contaminated with ERFX excreted by medicated

humans or domestic animals.

In summary, the present study suggests that the spread of

antimicrobial‑resistant E. coli in wild animals may be influenced by both of

anthropogenic and non‑anthropogenic factors. The low prevalence of

antimicrobial‑resistant E. coli in wild animals suggests that non‑a̲nthropogenic factors might have greater influence than anthropogenic ^ones such ^as human activities under

some circumstances. The

genotyping

results imply that host species, animal behaviors and genetic

diversity

in intestinal E. coli population ^are possible non‑anthropogenic

factors. Detection of ERFX‑resistant E. coli implies that antimicrobial ^use isone of the

anthropogenic factors. Further studies ^are needed ^to identify other contributing factors and analyze the influence of each factor in various envirorments, ranging from conserved ^{areas to} urban ^areas.

42

S UmRY

Antimicrobia1‑resistant E. coli ^are thought ^to have spread in wild animals through their contact with human activities. To elucidate the factors affecting the spread of antimicrobia1‑resistant strains in wild animals, E. coli from wild animals ^were screened for antimicrobial resistance and ^were characterized by

genotyping

^{in four areas}

in Japan where wild animals interact with humans. A total of 350 fTecal samples ^were

collected in 2008 and 2009 and 949 E. coli isolates ^were recovered from 219 samples.

One isolate from a sika deer was resistant ^to OTC, carrying the

tet(B)

^{gene. Four}

isolates from a raccoon dog were resistant ^to ABPC, NA and ERFX, carrying the blaTEM

gene. Four amino acid substitutions ^were observed in GyrA

(S83L, D87N)

and ParC

(S80I,E84V)

of E. coli isolates resistant ^to ABPC, NA and ERFX.

Genotyping

ofE.

coli isolates from wild animals suggests that predominant strains ^are speciflC ^tO host species. Detection of ERFX‑resistant strains suggests that antimicrobial ^use in human and veterinary medicine is one of the factors affecting the spread of

antimicrobia1‑resistant E. coli in wild animals. The host

specificity

of predominant E.

coli implies that E. coli from humans or domestic animals ^are unlikely ^to be transmitted to or to colonize wild animals. The

genotyping

results also suggest that predominant E.

coli strains ^{were more} specific ^to individuals than ^to host species in wild animals other than Japanese macaques. This implies that the risk of intra‑species transmission of E.coli might be diverse depending on host species. As ^a conclusion, the findings in this

study suggest that the spread of antimicrobial‑resistant E. coli in wild animals may be influenced by both of anthropogenic factors such ^as antimicrobial ^use and other factors

such ^as animal species.

Table 8. Antimicrobial susceptibility^tests used in this study.

Method Antimicrobialsb

(amount

^o,

concentration)

Disc difhsion method

Screening with M‑Ha agars

Broth microdilution method

ABPC

(10 LLg),

^CEZ

(30 pg),

^CTX

(30 Llg),

^CAZ

(30 pg),

^SM

(10 Llg),

^KM

(30 Ltg),

^GM

(10 Ltg),

^OTC

(30 pg),

^NA

(30 pg),

NFLX

(10 LLg),

^OFLX

(5 Llg),

^CP

(30 Llg),

^ST

(23.75/I.25 Llg)

ABPC

(32 pg/m1),

^CEZ

(32 pg/ml),

^SM

(32 pg/m1),

^OTC

(l6 pg/m1),

^NA

(32 Llg/ml),

^ERFX

(4 LLg/ml),

^CP

(32 pg/m1),

^TMP

(16 pg/ml)

ABPC, OTC, NA, ERFX

(0.0625‑128

pg/ml for all

agents)

a M‑H ^‑ Mueller‑Hinton

bABPC ^‑ ampicillin; CEZ ^‑ cefazolin; CTX ^‑ cefotaxime; CAZ ^‑ ceftazidime; SM ^‑ streptomycin; KM ^‑ kanamycin; GM ^‑ gentamicin; OTC ^‑ oxytetracycline; NA ^‑ nalidixic acid; NFLX ^‑ norfloxacin; OFLX ^‑ ofloxacin; CP ^‑ chloramphenico1; ST ^‑ sulfamethoxazole‑

trimethoprim; ERFX ^‑ enrofloxacin; TMP ^‑ trimethoprim

44

Table 9. Primers used in the analyses of antimicrobial resistance ^genes and genotyping Product

Gene Primer Primer sequence (5'‑3') conca (pM)

size

Re ference tet(A) let(A)‑F

tet (A)‑R let(B) ^let(B)‑F

let(B)‑R

let(C) ^let(C)‑F

tet(C)‑R

let(D) tet(D)‑F let(D)‑R let(M) ^let(M)‑F

let(M)‑R

blaPSE blaPSE‑F blaPSE‑R

bla SHV ^SHV‑F SHV‑R

blaTEM TEM‑C TEM‑H

bla OXA‑ ¹ 0XA‑F OXA‑R

gy7.A gyrA‑ F

gyrA‑R

gyrB gyrB ‑F

gyrB ‑R

parC parC‑F

parC‑R

parE parE‑F

parE‑R

chuA ChuA. 1

ChuA. 2

JjaA YjaA.¹ YjaA.2

TspE4.C2 TspE4C2.1 TspE4C2.2

GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG

TTG GTT ^{AGG GGC AAG TTT TG}

GTA ATG GGC CAA TAA CAC CG

CTT GAG AGC CTT CAA CCC AG

ATG GTC GTC ATC TAC CTG CC

AAA CCA ^TTA CGG CAT ^TCT GC

GAC CGG ATA CAC CAT CCA TC

GTG GAC AAA GGT ACA ACG AG

CGG TAA AGT TCG TCA CAC AC

TTT GGT TCC GCG CTA TCT G

TAC TCC GAG ^{CAC CAA ATC CG}

AGG ATT GAC TGC CTT ^TTT G

ATT TGC TGA TTT CGC TCG

ATC AGC AAT AAA CCA GC CCC CGA AGA ACG TTT TC

ATA TCT CTA CTG ^TTG CAT CTC C

Aju CCC TTC AAA CCA TCC

ACG TAC TAG GCA ATG ACT GG

AGA AGT CGC CGT CGA TAG AAC

CTC CTC CCA GAC CAA AGA CA TCA CGA CCG ATA CCA CAG CC

TGT ^ATG CGA TGT CTG AAC TG CTC ^AAT AGC AGC TCG GAA ^TA

TAC CGA GCT GTT CCT TGT GG GGC AAT GTG CAG ACC ATC AG

GAC GAA CCA ACG GTC AGG AT TGC CGC CAG TAC Cju AGA CA

TGA AGT GTC AGG AGA CGC TG ATG GAG AAT GCG TTC CTC ^AAC

GAG ^TAA TGT CGG GGC ^{ATT CA} CGC GCC AAC ^{AAA GTA TTA} CG

1 210 78

1

0.25 659

0.25

0.25 418

0.25

2 787

2

0.5 406

0.5

0.5 150

0.5

0.5 392

0.5

1 516

I

1.5 619

I.5

0.25 189

0.25

0.25 447

0.25

0.25 264

0.25

0.25 266

0.25

2 279

2

2 211

2

2 152

2

78

78 78

78 78

78 78

78 78

16 16

24 24

24 24

24 24

31 31

101 101

31 31

31 31

21 21

21 21

21 21

Table 10. Number offecal samples, number ofEscherichia coli isolates and the prevalence of antimicrobial‑resistantE. coli in each research site.

No. of fecal samples No. ofE. colt isolates

Species S ampling

methoda Total

̲p.s;;i::l;'%b,

Resistant(%c, ^{Total Resistant}

Rausu Sika deer (Cervus nljpon ) ^{Fecal collection 22 12} (54.5%) ¹(8.33%) ^{69 1}

culled 19 14 (73.7%) ^{0 52 0}

Hunted 4 3 (75.0%) ^{0 18}

Total 45 29 (64.4%) ¹(3.45%) ¹³⁹

Shimokita Japanese macaque

(Macacafuscata

⁾

Japanese serow

(Capricornis

_crispus)

Japanese hare (Lepus^brachyurus )

Raccoon dog (Nyctereutesprocyonoides)

Japanese black bear

(U7TuS thibetanus)

Red fox (Vulpes vulpes)

Japanese marten (Martes _melampus )

Unknown mammals

Copper pheasant (Syrmaticus soemmerringii)

Fecal collection Culled/Captured Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection

103 51

(49.5%)

19 8 (42.1%)

6 4 (66.7%)

6 3 (50.0%)

4 2 (50.0%)

1 1

(100%)

1 0

1 0

1 0

3 1(33.3%)

0 0 0 0

175 0

29 0

17 0

12 0

1(50.0%) ^{16 4}

0

0

4 0

0 0 0

4 0

145 70 (48.3%) ¹ (I.43%) ^{257 4}

a Fecal collection ^‑ Fecal samples ^on the ground ^were collected by searching ^on foot or by car.

culled, Culled/Captured ^‑ Fecal samples ^were collected from animals culled ^or captured for pest control.

Hunted ^{‑ Fecal} samples ^were collected from hunted animals.

Dead ^‑ Fecal samples ^were collected from dead animals by the roadside.

bpercentage ofE. coli‑positive samples in collected samples.

c percentage of samples carrying antimicrobial‑resistant E. coli isolates in E. coli‑positive samples.

Table ^lO (Continued)

No. offecal samples No. ofE. coli isolates

Species

S ampling

methoda rota.

̲p.s?;i::l;'%b,

Resistant (%c, ^{Total Resistant}

Japanese macaque (M.

fuscata

⁾

Deer (C. nllpon) ^/serow (C. crispus)

Japanese hare (L.brachyurus)

Wild boar (Sus

scrofa)

Raccoon dog (N.procyonoides)

Red fox (V. vulpes)

Japanese marten (M. melampus)

Japanese squirrel (Sciurus lis)

Japanese giant flying squirrel (Petaurista^leucogenys )

Japanese black bear (Ursus thibetanus)

Nutria (Myocastor coypus )

Japanese badger

(Meles

anakuma

)

Unknown mammals Ileron (Ardeidae)

Northern Goshawk (AcclPitergentilis )

Copper pheasant (S.soemmerringii)

Unknown birds

Fecal collection Fecal collection Fecal collection Hunted

Fecal collection Dead

Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection Dead

Dead

Fecal collection Fecal collection Fecal collection Fecal collection Fecal collection

26 25 (96.2%)

41 27 (65.9%)

9 0

8 7

(87.5%)

3 3 (100%)

1 1(100%)

4 4 (100%)

4 3 (75.0%)

2 0

1 0

1 0

1 0

1 0

6 3

(50.0%)

2 2 (100%)

1 0

2 2 (100%)

2 1 (50.0%)

0 0 0 0 0

0 0

0 0

154 0

135 0

0 28 13 3 16 12 0 0 0 0 0 12

8 0 8 4

0 0 0 0 0

0 0

0 0

Total 1 15 78 (67.8%) ⁰

a Fecal collection ^‑ Fecal samples ^on the ground ^were collected by searching ^on foot or by car.

Table 10 (Continued)

No. of fecal samples No. ofE. coli isolates

Species

S ampling

methoda Total

̲p.s?;i::I;'%b,

Resistant (%c, ^{Total Resi stant}

Yakushima Japanese macaque (M,

fuscata

⁾

Sika deer (C. nilPOn)

Raccoon dog (N.procyonoides)

Fecal collection Fecal collection Fecal collection Dead

30 29 (96.7%) ⁰

13 12 (92.3%) ⁰

1 1 (100%) ⁰

I 0 0

109 0

48 0

3 0

0 0

Total 45 42 (93.3%) ^{0 160 0}

Total (All sites) ³⁵⁰

219(62.6%) 2(0.91%)

⁹⁴⁹

a Fecal collection ^‑ Fecal samples ^on the ground ^{were collected} by searching ^on foot or by car.

culled, Culled/Captured ^‑ Fecal samples ^were collected from animals culled ^or captured for pest control.

Hunted ^‑ Fecal samples ^were collected from hunted animals.

Dead ^‑ Fecal samples ^were collected from dead animals by the roadside.

bpercentage ofE. coli‑positive samples in collected samples.

c percentage of samples carrying antimicrobial‑resistant E. coli isolates in E. coli‑positive samples.

Table 1 1. Antimicrobial resistance patterns and resistance gene profiles of antimicrobia1‑resistantEscherichia coli isolates from wild animals.

Resistance gene profile

swain"o.

Site(Sample"o.)

Hostspecies Resishncepauern("IC'a _{let bla}

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