Multi-drug resistant Escherichia coli isolated from canine feces in a public park in Quito, Ecuado by Actuaciencia

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Title: Multi-drug resistant Escherichia coli isolated from canine feces in a public park in

Quito, Ecuador

Running title: MDR E. coli in a public park

4 5

David Ortega-Paredes1,2,3*, Marco Haro3,4, Paula Leoro-Garzón3,4, Pedro Barba5, Karen

Loaiza3, Francisco Mora1,6, Martha Fors1, Christian Vinueza-Burgos2, Esteban Fernández-

Moreira1.

Quito-Ecuador

Carrera de Medicina. Facultad de Ciencias de la Salud. Universidad de las Américas,

los Antibióticos. Facultad de Medicina Veterinaria y Zootecnia. Universidad Central del

Ecuador, Quito, Ecuador

Life Science Initiative, lsi-ec.com, Quito-Ecuador

Laboratorio Clínico e Inmunológico INMUNOLAB, Quito-Ecuador

Carrera de Ingeniería en Biotecnología. Facultad de ingeniería en Ciencias Agropecuarias

y Ambientales. Universidad Técnica del Norte, Ibarra-Ecuador

Unidad de Investigación de Enfermedades Transmitidas por Alimentos y Resistencias a

Hospital General del Sur de Quito (IESS), Quito-Ecuador

18 19 20

*Corresponding author at: Carrera de Medicina. Facultad de Salud. Universidad de las

Américas. Vía a Nayón, Quito 170124. daortegap@gmal.com;

david.ortega.paredes@udla.edu.ec

ABSTRACT

Objectives: We focused on estimating the prevalence of extended-spectrum-Î˛-lactamase

(ESBL), pAmpC, carbapemenases, and mcr-1 producing Escherichia coli in canine feces from

a public park in Quito, Ecuador.

Methods: We performed phenotypic and genotypic characterization of E. coli isolated from

50 canine feces samples recovered from a city park in Quito, Ecuador. Additionally, a

multiple-choice survey was conducted among 50 dog owners.

Results: Twenty out of 50 samples (40%) presented E. coli resistant to ceftriaxone; 23 E. coli

isolates were recovered for further analysis. All of the isolates showed the phenotype for

multi-drug resistance (resistance to â&#x2030;Ľ 3 antibiotic families). Resistance to carbapenems,

tigecycline, and amikacin were not registered. No major clonal relatedness was observed

among the resistant isolates. ESBL alleles blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were the

most common. Two isolates harbor blacmy-2 gene and one isolate harbor both mcr-1 and

blaCTX-M-65 genes. Statistical analysis showed that older people were more conscious of

collecting and disposing of dogâ&#x20AC;&#x2122;s feces than subjects younger than 35 years old (p < 0.05).

Conclusions: Our finding of multidrug resistant (MDR) E. coli in dog feces found in a city park

illustrates the importance of analyzing canine feces in public settings (e.g., parks,

playgrounds) as part of MDR E. coli surveillance programs. Additionally, our research might

be a sentinel sampling method of study to gain a better understanding of community

sources of antibiotic-resistant

interfaces.

Enterobacteriaceae

at human-animal-environment

Keywords: pAmpC Î˛-lactamases; Ecuador; Escherichia coli; ESBL; canine feces; mcr-1; multi-

drug resistance; public park.

1. Introduction

Worldwide, the presence of animal feces in the ground of urban areas is a significant public

health problem, due to the presence of different microorganisms that can be transmitted

to humans [1]. This concern is increasing with the growing pet population and free roaming

animals in large cities like Quito in Ecuador [2,3]. This increment has augmented the

probability of contact between animals and humans, it has heightened the exposure risk to

different pathogens, including multi-drug resistant (MDR) Escherichia coli [1]. MDR E. coli

has been isolated from humans, animals, and the environment worldwide [4]. The most

important resistance mechanisms in these strains are extended-spectrum-Î˛-lactamase

(ESBL),

phosphoethanolamine-lipid A transferases (mobile colistin resistance, mcr), which lead to

therapy failure.

Despite the studies on ESBL, pAmpC, carbapenemases, and mcr producing E. coli from

different sources, the routes of transmission are missing, and the epidemiology of these

strains is poorly understood. So far, the prevalence and the potential for transmission of

MDR E. coli between companion animals and humans are unknown. The aim of this study

was to estimate the prevalence of ESBL, pAmpC, blaKPC, and mcr-1 genes in ceftriaxone-

resistant E. coli isolated from canine feces in a city park in Quito, Ecuador.

2. Materials and methods

2.1. Study design

We performed an exploratory, observational study to determine the prevalence of

ceftriaxone-resistant E. coli in canine feces from a city park in our city. Quito, the capital city

plasmid-mediated

pAmpC

Î˛-lactamases,

carbapenemases,

and

of Ecuador, is located in the Andean Region of Ecuador (0°13′07″S and 78°30′35″W), at an

altitude of 2,810 meters above sea level, with a population of 1´911.966 [5]. In this study,

we establish a transect of 1 km2 in a lineal park in the southern part of the city (-0.262678,

-78.538390; -0.266270, -78.550295), where families walk their dogs, people and children

play and do exercise, street food is sold, and where there are no facilities for the collection

of canine feces. The collection of feces is regulated by two local ordinances [20, 21].

2.2. Sampling and isolation

We collected 50 samples in August 2017. Samples were taken from the top of fresh feces in

order to avoid cross contamination from environmental bacteria of the ground. Samples

were collected in sterile plastic flasks, and sent to be processed in a laboratory, up to an

hour after collection. Samples were plated, using a sterile swab, in MacConkey agar (Becton,

Dickinson and Company, France) supplemented with 3 µg of ceftriaxone (CRO) and

incubated at 35°C for 18-20 hours. Plates growing red/pink colonies were registered as

positive. All different E. coli-like colonies were spiked and isolated in the same medium. A

full loop of bacteria was mixed with trypticase soy broth (TSB, Becton, Dickinson and

Company, France) + 40% glycerol (Himedia, France) and stored at -20°C for further analysis.

2.3. Identification and susceptibility testing

Species confirmation was performed using biochemical tests: triple sugar iron (TSI),

Simmons citrate test, urease production, tryptophan reduction (indole), methyl-red test

(RM) and Voges Proskauer (VP) test and confirmed by matrix-assisted laser desorption

ionization time-of-flight mass spectrometry (MALDI-TOF) analysis in a Microflex LT MALDI-

TOF spectrometer (Bruker Daltonics, Inc.).

To identify ESBL, pAmpC, or carbapenemase producing phenotypes, E. coli isolates were

screened with double disk synergy using cefotaxime (30 µg), cefotaxime/clavulanic acid

(30/10 µg), ceftazidime (30 µg), ceftazidime/clavulanic acid (30/10 µg), and susceptibility to

cefepime (30 µg), imipenem (10 µg), and cefoxitin (30 µg). Antibiotic resistance profiles

were carried out using the disk diffusion method with aztreonam (30 µg), imipenem (10 µg),

tetracycline (30 µg), tigecycline (10 µg), doxycycline (30 µg), ciprofloxacin (5 µg), nalidixic

acid (30 µg), norfloxacin (5 µg), levofloxacin (5 µg), gentamicin (10 µg), netilmicine (30 µg),

amikacin (30 µg), fosfomycin (200 µg), nitrofurantoin (300 µg), chloramphenicol (30 µg),

trimethoprim/sulfamethoxazole (1,25/23,75 µg), and azithromycin (15 µg) (Becton,

100

Dickinson and Company, France). BD Phoenix™ Automated Microbiology System (France)

101

was used for colistin. Results were interpreted following the Clinical and Laboratory

102

Standards Institute (CLSI, 2017) recommendations.

103

2.4. Genotypic characterization

104

Resistance genes were identified using polymerase chain reaction (PCR) protocols

105

described elsewhere: blaCTX-M Group 1 [7], blaCTX-M Group 2 [8], blaCTX-M Group 8 [9], blaCTX-

106

M Group

107

controls were included in each reaction. The phylogenetic group was also identified [13].

108

All amplicons were sequenced in both strains using the dideoxy sequencing method.

109

Sequences were edited and analyzed in Geneious R10 using the database of ResFinder-3.0

110

[14]. Clonal analysis was carried out using the BOX-PCR method described elsewhere [15],

111

and cluster analysis was completed in GelCompar II version 6.6.11 (Applied Maths).

9 [10], for blaKPC [11], and colistin resistant mcr-1 [12]. Positive and negative

112

Fragments between 200 bp and 1,500 bp in size were included in the analysis using the

113

UPGMA model with DICE coefficient.

114 115

2.5. Survey

116

Multiple-choice questionnaires were completed with face-to-face interviews conducted

117

with 50 dog owners who were walking their dogs in the park (after observing whether they

118

pick up after their dogs or not). Dog owners were asked several questions with the intention

119

of assessing their dog-owner habits, in particular their attitudes concerning the disposal of

120

their dog’s feces. The questionnaire included a total of 3 questions focused on establishing

121

the participant’s attitude and behavior with respect to dog fouling in the park and what

122

factors (e.g., availability of bags and bins, fines, and reasons) influence the decision to clean

123

up their dog’s waste. In addition, participants were asked to rate the importance of stated

124

factors that have influenced their behavior in relation to clearing up their dog feces (e.g.,

125

claims from a member of the public). We also gathered qualitative information from the

126

participants’ points of view regarding waste disposal and personal opinions about the

127

reasons for not collecting feces in the park. We did not collect information about whether

128

the dogs present in the parks live in a home/dwelling with at least one other animal, the

129

frequency of visiting the park, or regarding animal healthcare. The survey was adapted from

130

the Dog Park Survey made by the Parks Advisory Commission of the City of Ann Arbor,

131

Michigan [16]

132

2.6. Statistical Analysis

133

We calculated absolute and relative frequencies for qualitative variables of the survey.

134

Fisher's exact test was used to compare proportions, and p value <0.05 was considered as

135

statistically significant. Confidence interval analysis was carried out using R, V. 3.2.5.

136

3. Results

137

3.1. Isolation and phenotypic characterization

138

Twenty out of 50 samples showed the growth of E. coli-like colonies on antibiotic-

139

supplemented McConkey agar plates with a prevalence of 40% (CI: 39.74; 40.55). From the

140

20 samples, 23 isolates were selected and confirmed as E. coli, one per sample except for

141

sample 26, in which colonies 26Apl, 26Bpl, and 26Cpl were included and sample 49, in which

142

colonies 49Apl and 49Bpl were included). All 23 isolates were submitted to the screening

143

for β-lactamase production. Twenty-one isolates were positive for ESBL production, two to

144

pAmpC β-lactamases production, and there were no isolates suspicious to carbapenemases

145

production. All isolates were resistant to cefotaxime and tetracycline. We registered a high

146

percentage of resistance to ceftazidime, aztreonam, cefepime, doxycycline, ciprofloxacin,

147

nalidixic acid, norfloxacin, levofloxacin, and trimethoprim-sulfametoxazole, less than 50%

148

of resistance to gentamicin, netilmicin, fosfomycin, chloramphenicol, cefoxitin, and

149

azithromycin, and none of the isolates were resistant to imipenem, tigecycline, or amikacin.

150

All of the isolates were MDR (resistant to ≥ 3 families of antibiotics). One isolate presented

151

the mcr-1 gene and was resistant to colistin MIC = 4; ampicillin MIC > 16, ceftriaxone MIC >

152

32, cefuroxime MIC > 16, ciprofloxacin MIC > 2 and levofloxacin MIC > 4 (Figures 2 and 3).

153

3.2. Genetic characterization

154

No major clonal relatedness was observed among the resistant isolates. The phylogroup B1

155

was the most prevalent among the isolates, followed by A, D, and B2. The blaCTX-M Group 1

156

genes were the most prevalent (43.5%, 10 isolates), represented by the variants blaCTX-M-15

157

(17.4%, 4 isolates), blaCTX-M-55 (17.4%, 4 isolates), and blaCTX-M-3 (8.7%, 2 isolates), followed

158

by blaCTX-M Group 9 (26%, 6 isolates), with the variants blaCTX-M-65 (13%, 3 isolates), blaCTX-M-

159

14 (4,3%, 1 isolate), blaCTX-M-27 (1 isolate) and blaCTX-M-106 (1 isolate), and blaCTX-M Group 2, with

160

the variant blaCTX-M-2 (8.7%, 2 isolate). Additionally, one isolate (32 pl) presented the hybrid

161

blaCTX-M-123. The blaCTX-M Group 8 and blaKPC were not detected. One isolate (19 pl) presented

162

two blaCTX-M genes (blaCTX-M-2 and blaCTX-M-3). Two isolates (8.7%) presented the blaCMY-2 gene.

163

Finally, the isolate 4pl harbor blaCTX-M-65 and mcr-1 genes (Figure 2).

164

3.3. Owners survey results

165

The survey was carried out in Spanish, questionnaire in English is in supplementary data 1:

166

We collected answers from 50 dog owners. The mean age of owners was 36.6 years, with a

167

standard deviation of 13.6 years. Fifty-eight percent of the interviewees were men. Most of

168

the owners collected the fresh feces of their dogs (86%), a tendency that was more

169

accentuated in men; however, we did not find any significant statistical difference between

170

the habits of men and women in picking up feces.

171

The mean age of owners less than 35 years old was 25.6 years, with a standard deviation

172

(SD) of 6.2 years, while older subjects had a mean age of 47.7 years and SD of 8.2 years.

173

There are significant statistical differences between these two groups regarding collecting

174

and disposing of feces (p<0.05). Older people were more conscious about these tasks than

175

subjects younger than 35 years. From those to collect the waste of their dog, approximately

176

60% do it always, while the rest do it sometimes. (p<0.05). There were no statistically

177

significant differences in the rest of the variables explored.

178

There was a high percentage of participants who collected the feces because of hygienic

179

reasons. Also, the possibility of getting a fine was stated by the majority as a good reason

180

to collect the feces of their dogs. The leading excuse for not properly disposing of the feces

181

was the lack of availability of bins and bags (Table 1).

182

From those who do not collect the feces of their dogs we found that 71.4% did not collect

183

because of disgust or lack of bags or bins. (Figure 3). Forty-two percent do not care whether

184

they pick up the dogâ&#x20AC;&#x2122;s feces or not.

185

Regarding qualitative information, some participants expressed not having knowledge

186

about ordinances [20, 21] related to feces disposal, while others think that there is a lack of

187

personnel to enforce ordinances, and they also expressed also need for more educational

188

campaigns related to management of animal feces disposals.

189 190

4. Discussion

191

This is the first study performed to assess canine fecal contamination and resistance in an

192

urban park and dogs frequenting such a park in Ecuador. In most countries, the overall

193

number of antimicrobials used for companion animals is not consistently measured.

194

However, antimicrobials used in human and veterinary hospitals are almost identical. In

195

Ecuador, there is no data of veterinary prescription of antibiotics. Nevertheless, in our

196

experience, the most commonly prescribed antimicrobials in small animals are amoxicillin,

197

fluoroquinolones, third-generation cephalosporins, and tetracyclines. These prescribing

198

trends have been described elsewhere [17, 18]. Antimicrobial prescription has received

199

little attention in veterinary clinical settings, regarding the augmented bacterial resistance

200

worldwide. The role of pets in the dissemination of antimicrobial resistance has been given

201

little attention when compared with that of food animals [19]. Companion animals are

202

thought to be a reservoir of resistant bacteria and the risk of spread to humans is facilitated

203

through their feces. In a study performed by Ferreira et al. (2017) [20], they found that

204

almost all dog owners (94.1%), claimed to collect their dog's feces. In our study, this figure

205

was smaller (83.3%). This percentage may be overestimated since the survey was not

206

anonymous.

207

Although there were dog feces bag dispensers at the park, all of them were without

208

plastic bags. Dog owners who picked up the feces carried their own plastic bags. The

209

collection of pet waste is regulated in Quito by two ordinances. The ordinance of the

210

Metropolitan Council of Quito # 48 in articles 6 and 30 of the Municipal Code For The

211

Metropolitan District Of Quito, Municipal Ordinance 1, specifically the article 357.2 [21,

212

22], provides a penalty for not picking up the dogs feces. Nevertheless, these ordinances

213

are not well known by the owners of animals in the city and poorly implemented by the

214

local police. The fact that older people are more aware of hygiene than younger people

215

(Table 1) suggests that more educational campaigns are needed. These educational

216

campaigns should be related to management of animal feces disposal in order to avoid

217

the spread of MDR E. coli.

218

To date, the prevalence of third generation cephalosporin-resistant E coli in dog feces from

219

public parks has been poorly described. In our work, we reported a 40% (20/50) prevalence,

220

which is much higher than the prevalence reported for other public spaces (1.9% - 4/209),

221

such as public gardens in Denmark [23]. On the other hand, there are several studies on

222

ESBL-E. coli prevalence in healthy dogs that have been reported prevalence from 45% (9/20)

223

in the Netherlands [24], 17% (9/53) in Mexico [25], to 7,2% (5/102) in Algeria [26]. To the

224

best of our knowledge, there are no reports of the Andean region in the indexed literature.

225

Despite using direct plating on the selective MacConkey agar plate method, without an

226

enrichment step, which could lead to the loss of positive samples with low counts, as

227

reported elsewhere [24]. However, the prevalence (40%) of samples carrying E. coli

228

resistant to third-generation cephalosporins in our study is worrisome for a public area.

229

Genes belonging to the blaCTX-M family are the most frequently reported in human, animal,

230

and environment ESBL-E coli isolates [27], and blaCMY-2 is the most frequently identified

231

pAmpC gene worldwide [28]. The same pattern has been identified in canine fecal samples,

232

where blaCTX-M-1, blaCTX-M-15 (blaCTX-M-group 1), and blaCMY-2 have been frequently described [24,

233

25, 26].

234

In this study, we identified blaCTX-M genes belonging to groups 1, 2, and 9. In the blaCTX-M-group

235

236

prevalent worldwide, and blaCTX-M-55 is increasing in prevalence in several locations [29, 30].

237

The most prevalent blaCTX-M-group 9 was blaCTX-M-65, previously described in our location [31].

238

Our screening for carbapemenase production was based in the sensitivity to imipenem.

239

However, there have been reports of enterobacteriaceae strains harboring blaKPC genes

240

sensitive to imipenem. Additionally, in Ecuador, blaKPC is the most prevalent gene associated

241

with carbapenem-resistant in Enterobacteriaceae [11]. Therefore, we screen all the isolates

variants blaCTX-M-15 and blaCTX-M-55 were the most prevalent. blaCTX-M-15 is the most

242

for this gene [32]. However, we did not register isolates harboring blaKPC. Nevertheless,

243

carbapenemases-producing E. coli isolated from dog feces has been reported previously

244

[33].

245

Colistin is the last resort antibiotic against extensively resistant Gram-negative bacteria.

246

Plasmid-mediated colistin-resistant gene mcr-1 was described in E. coli isolates from food

247

animals and humans in November 2015 [34]. In Ecuador, the mcr-1 gene was reported in

248

2016 in a human E. coli isolate from peritoneal fluid [12]. To date, the mcr-1 gene has been

249

reported worldwide from a variety of sources [35]. However, there are few reports of E. coli

250

harboring mcr-1 isolated from dogs. In this study, the isolation of E. coli harboring mcr-

251

1/blaCTX-M-65 from canine feces in a city park, opens the possibility of further studies to

252

determine whether there is a potential transmission of these strains to humans as it was

253

reported previously [36, 37]. Additionally, the high genotypic diversity shown by the clonal

254

analysis, suggests a variety of genetic backgrounds of resistant determinants present in

255

MDR E. coli in dogs.

256

A variety of reports support the transmission of E. coli between dog feces, dogs, and

257

humans. Schaufler et al. [38] reported dog feces around veterinary facilities as a non-point

258

infection source of ESBL-producing E. coli for both animals and humans. Dog feces have

259

been identified as a vector for dissemination of ESBL-E. coli within urban areas [23]. As mcr-

260

1â&#x20AC;&#x201C;producing E. coli in dogs can be transferred between companion animals and humans

261

[36, 39], we wanted to know if dog feces in a Ecuadorian park could be a source of MDR

262

bacteria for humans. Finally, cross-transmission of resistant E. coli can easily occur between

263

owners and non-owners [40]. Based on this evidence, the dissemination of these bacteria

264

from the dog feces in the park to humans is plausible. This hypothesis must be examined

265

more thoroughly to establish the real implication of dog feces in city parks over human and

266

pet heath.

267 268

5. Conclusion

269

Although people consider public parks excellent recreational areas for their family and dogs,

270

our results highlight the potential of urban parks as a reservoir and source of transmission

271

of MDR bacteria. Additionally, actions like improving the availability of disposal bags, dog

272

ownerâ&#x20AC;&#x2122;s education, and fines could help to limit their dissemination. Further studies are

273

needed to evaluate the role of dog feces contamination in public environments, as a

274

dissemination pathway of bacterial resistance. The characterization of isolates from canine

275

feces in public parks might be a sentinel sampling method to gain a better understanding of

276

community sources of antibiotic-resistant enterobacteriaceae at human-animal-

277

environment interfaces.

278 279 280 281

Acknowledgments

282

of this work was presented in Quito at the Jornadas Nacionales de BiologĂa in September

283

2017.

The authors would thank to Elvis Romero for his assistance in the project development. Part

284 285

Funding: Microbiological and molecular analysis were supported by UDLA project number:

286

MED.EFM.18.08, UNIETAR, LSI, INMUNOLAB and Hospital General del Sur de Quito (IESS).

287 288

Competing interests: None declared

289 290

Ethical approval: Not required

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Figure 1. Resistance patterns and genetic characterization. CTX, cefotaxime; CAZ; ceftazidime; ATM, aztreonam, FEP; cefepime, FOX; cefoxitin, IMP; imipenem, TE; tetracycline, TGC; tigecycline, DO; doxycycline, CIP; ciprofloxacin, NA; nalidixic acid, NOR; norfloxacine, LEV; levofloxacin, GEN; gentamicin, NET; netilmicin, AK; amikacin (30 µg), fosfomycin (200 µg), nitrofurantoin (300 µg), chloramphenicol, SXT; trimethoprim/sulfamethoxazole, AZM; azithromycin. Figure 2. Resistance profiles CTX, cefotaxime; CAZ; ceftazidime; ATM, aztreonam, FEP; cefepime, FOX; cefoxitin, IMP; imipenem, TE; tetracycline, TGC; tigecycline, DO; doxycycline, CIP; ciprofloxacin, NA; nalidixic acid, NOR; norfloxacine, LEV; levofloxacin, GEN; gentamicin, NET; netilmicine, AK; amikacin (30 µg), fosfomycin (200 µg), nitrofurantoin (300 µg), chloramphenicol, SXT; trimethoprim/sulfamethoxazole, AZM; azithromycin. Figure 3. Causes for not collecting dogs’ feces.

Table 1. Statistical analysis of attitudes of dog owners regarding the disposal of dog feces survey.

Table 1

CHARACTERISTICS

COLLECTING FECES Yes n=43

TOTAL

p value*

Non=43

No.

â&#x2030;¤35 years old

46.5

85.7

52.0

35 years old

53.5

14.3

48.0

Always

60.5

0.0

52.0

Sometimes

39.5

71.4

44.0

Never

0.0

28.6

4.0

Male

55.8

71.4

58.0

Female

44.2

28.6

42.0

Group of age 0.05

Collection frequency 0.01

Gender 0.48

Claims from a member of the public Yes

20.9

42.9

24.0

79.1

57.1

76.0

Yes

79.1

85.7

80.0

20.9

14.3

20.0

Yes

32.6

57.2

36.0

67.4

42.8

64.0

Yes

62.8

71.5

64.0

37.2

28.5

36.0

Yes

51.2

42.8

50.0

48.8

57.2

50.0

Yes

51.2

42.8

50.0

48.8

57.2

50.0

0.20

Hygienic reasons 0.68

Because of children 0.20

Availability of bins 0.65

Availability of bags 0.68

Fines 0.68

*Fisher exact Test

Figure 1

Click here to access/download;Figure;Figure 1.jpg

Figure 2

Click here to access/download;Figure;Figure 2.jpg

Figure 3

Click here to access/download;Figure;Figure 3.jpg