Antimicrobial resistance in freshwater Plesiomonas shigelloides isolates: Implications for environmental pollution and risk assessment

Journal Pre-proof Antimicrobial resistance in freshwater Plesiomonas shigelloides isolates: Implications for environmental pollution and risk assessment Temitope Cyrus Ekundayo, Anthony I. Okoh PII:

S0269-7491(19)32028-7

DOI:

https://doi.org/10.1016/j.envpol.2019.113493

Reference:

ENPO 113493

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Environmental Pollution

Received Date: 17 April 2019 Revised Date:

23 August 2019

Accepted Date: 24 October 2019

Please cite this article as: Ekundayo, T.C., Okoh, A.I., Antimicrobial resistance in freshwater Plesiomonas shigelloides isolates: Implications for environmental pollution and risk assessment, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113493. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Antimicrobial resistance in freshwater Plesiomonas shigelloides isolates: implications

2

for environmental pollution and risk assessment

3 Temitope Cyrus Ekundayo1,2,3* and Anthony I. Okoh1,2

4 5 1

6

SAMRC Microbial Water Quality Monitoring Centre, University of Fort Hare, Alice,

7 8

Eastern Cape, South Africa 2

Applied and Environmental Microbiology Research Group, Department of Biochemistry and

9 10

Microbiology, University of Fort Hare, Alice, Eastern Cape, South Africa 3

Department of Biological Sciences, University of Medical Sciences, Ondo City, Ondo State,

11

Nigeria

12 13

*Corresponding author (T.C. Ekundayo). Email: [email protected]

14 15

Abstract

16

Antibiotic resistance is known to impact treatment efficiency of Plesiomonas infections

17

negatively with fatal outcomes. This study investigated antibiogram fingerprint of P.

18

shigelloides (n = 182) isolated from three South Africa rivers using the disc diffusion

19

technique. Environmental pollution and analogous health risk (given infections) that could

20

associate with the freshwaters and empirical treatment of Plesiomonas were assessed using

21

Antibiotic Resistance Index (ARI) and Multiple Antibiotic Resistance Indices (MARI),

22

respectively. Thirteen EUCAST recommended (ERAs) and eleven non-recommended

23

antibiotics (NAs) used as first line agents in the treatment of gastroenteritis and

24

extraintestinal infections were tested. Resistance against ERAs decreased from cefoxitin

25

(37.91%), cefuroxime (35.17%), cefepime (31.87%), ceftriaxone (29.67%), ciprofloxacin

26

(18.13%),

27

ertapenem (4.95%), norfloxacin (4.40%), levofloxacin (2.75%), meropenem (1.10%) to

trimethoprim-sulfamethoxazole

(10.44%),

piperacillin/tazobactam

(8.79%),

28

imipenem (0.55%). The isolates had higher resistance (≥36.07%) against NAs but were

29

susceptible to amikacin (67.58%), gentamycin (73.08%), and tetracycline (80.77%). MARI of

30

the isolates were significantly different between ERAs and NAs (P-value < 0.05) and had an

31

average of 0.17±0.18 and 0.45±0.13, respectively. About 33.87% and 95.63% of the isolates

32

had MARI value from 0.23 − 0.62 and 0.27 − 0.82 to ERAs and NAs, respectively. Also,

33

ERAs-based and NAs-based ARI across sampling units showed significantly different (P-

34

value < 0.05) means of 0.18±0.09 and 0.46±0.05, respectively. MARI attributed low risk of

35

empirical treatment to recommended antibiotics but higher risk to non-recommended

36

antibiotics. Model estimated successful and unsuccessful empirical treatment of infections

37

risks due to resistance in the isolates using recommended antibiotics as 65.93% and 34.07%,

38

respectively; 1.65% and 98.35% in the case of non-recommended antibiotics, respectively.

39

ARI based on recommended antibiotics identified potential environmental pollutions in a

40

number of sites. Resistance in freshwater P. shigelloides especially against cephalosporin,

41

quinolones and fluoroquinolones is distressing and might suggests high pollution of the

42

freshwaters in the Eastern Cape Province.

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Keywords: Plesiomonas shigelloides; antimicrobials resistance index; risk assessment;

44

public health.

45

Capsule: the study revealed multi-resistance of P. shigelloides against last resort antibiotics;

46

identified pollution risk in 8 sites and greater risk of fatal outcomes of empirical treatment of

47

infection by non-recommended antibiotics.

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1. Introduction

49

Antimicrobial resistance is one of the major clinical and veterinary challenges worldwide.

50

More disturbing is the resistance of freshwater microorganisms against important antibiotics

51

contrary to their antibiotics sensitive nature. The aquatic environments are now regarded as

52

repositories of antibiotic-resistant bacteria and their resistance genes (Korzeniewska et al.,

53

2013; Li et al., 2014; Leonard et al., 2015; Xu et al., 2015; Calero-Cáceres and Muniesa,

54

2016). Basically, the aquatic environments are open to anthropogenic pollutions (Zhu et al.

55

2017; Shao et al. 2018; Antti et al. 2018; Jiang et al. 2018) from a wide range of influences

56

including storm water/agricultural wastes or surface run-offs (Ahmed et al. 2018); wastewater

57

treatment plant (Rizzo et al. 2014; Hembach et al. 2017; Proia et al. 2018a; Proia et al.

58

2018b); hospital effluents (Harnisz and Korzeniewska 2018); industrial wastes (Karkman et

59

al. 2017);agricultural runoffs from slaughterhouses (Wan and Chou 2015; Fang et al. 2018);

60

effluents from landfills, floodwaters, and recreational functions. Many of these are heavily

61

laden with antibiotics residues, pesticides, herbicides, biocides, pharmaceuticals, textile dye

62

/organic compounds, heavy xenogenetic elements, disinfectants, antibiotic resistant bacteria

63

and antibiotic resistant genes (Jiao et al., 2017; Karkman et al., 2017; Fang et al., 2018; Guo

64

et al., 2018; Harnisz and Korzeniewska, 2018; Jiang et al., 2018; Li et al., 2018; Shao et al.,

65

2018; Zhang et al., 2018).

66

Plesiomonas shigelloides, the only oxidase-positive member of the Enterobacteriaceae, is

67

one of the freshwater- and aquatic resources-borne microorganisms (Wadström and Ljungh,

68

1991; Brenner et al., 2005). It is a Gram-negative rod, motile and non-spore-forming bacteria

69

recognized as a water- and food-borne pathogen and recently grouped into the order

70

Enterobacterales (Brenner et al., 2005; Meng et al., 2012; Santos et al., 2015). P. shigelloides

71

has been isolated from mollusks, crustaceans, reptiles and fishes as well as birds, amphibians,

72

and mammals (Oxley et al., 2002; Huber et al., 2004; Salerno et al., 2007; Alexander et al.,

73

2016; Pan et al., 2017). Mostly, regional distribution of the bacteria is often attributed to the

74

tropics and the subtropics including Southeast Asia, Africa and South America in particular

75

(Arai et al., 1980; Obi et al., 1995; Canosa and Pinilla, 1999; Shigematsu et al., 2000; Gibotti

76

et al., 2000; Tseng et al., 2002; Wong et al., 2000; Maluping et al., 2005;Chen et al., 2013;

77

Nwokocha and and Onyemelukwe, 2014). Notwithstanding, P. shigelloides has been isolated

78

in the cold and temperate regions of Sweden ( Krovacek et al., 2000; Gonzalez-Rey et al.,

79

2003), Netherlands, Serbia, Hungary and Czech Republic ( Medema and Schets, 1993;

80

Aldová et al., 1999; Bardon, 1999; Petrušić et al., 2018).

81

P. shigelloides is very important in human and veterinary medicines as several cases of

82

gastrointestinal and extraintestinal infections have been linked to the pathogen (Ampofo et

83

al., 2001; Auxiliadora-Martins et al., 2010b; Ozdemir et al., 2010; Bonatti et al., 2012; Xia et

84

al., 2015; Bowman et al., 2016). Gastrointestinal infections from P. shigelloides are

85

sometimes self-limiting (Holmberg and Farmer, 1984; Holmberg et al., 1986) but duration of

86

illness and treatment of (extraintestinal) infections often take up to 3 ─ 56 days (Ampofo et

87

al., 2001; Auxiliadora-Martins et al., 2010b; Bonatti et al., 2012; Bowman et al., 2016; Xia et

88

al., 2015). Many deaths have also resulted from Plesiomonas extraintestinal infections,

89

especially among children ( Nolte et al., 1988; Billiet et al., 1989; Terpeluk et al., 1992; Chen

90

et al., 2016; Xia et al., 2015). The clinical importance of P. shigelloides continues to grow in

91

recent times (Bonatti et al., 2012; Pfeiffer et al., 2012; Bowman et al., 2016; Ding et al.,

92

2015; Novoa-Farías et al., 2016; Patel et al., 2016; Pence, 2016; Hustedt and Ahmed, 2017a,

93

2017b). Other public health issues traced to P. shigelloides include food- and water-borne

94

outbreaks (Graciaa et al., 2018; Houten et al., 1998; Wouafo et al., 2006), and disease

95

outbreak in farmed Percocypris pingi in China (Pan et al., 2017).

96

Antibiotic therapy remains one of the primary instruments in infection control and

97

management. However, antibiotic resistance is known to impact treatment efficiency of

98

Plesiomonas infections negatively. Treatment failures and several deaths from Plesiomonas

99

infections have been attributable to resistance and wrong prescription of antibiotics (Pathak et

100

al., 1983; Terpeluk et al., 1992). Studies have revealed survival in neonatal extraintestinal

101

infections caused by P. shigelloides involved treatment with at least one or combination of

102

antibiotics from cephalosporins, carbapenems, aminoglycosides and quinolones (Eason and

103

Peacock, 1996; Riley et al., 1996; Ozdemir et al., 2010). More worrisome, P. shigelloides is

104

not among first-line pathogens routinely assayed in clinical laboratory and in part, share

105

some clinical manifestations with other pathogens, which often lead to its misdiagnosis (Chen

106

et al., 2013) and consequently increasing health risk due to Plesiomonas infections.

107

In this study, antibiogram fingerprint of P. shigelloides isolates from rivers in the Eastern

108

Cape Province, South Africa was investigated. The study modelled environmental pollution

109

risk (EPR) and empirical treatment risk (ETR) of Plesiomonas (given infections) with

110

recommended and non-recommended antibiotics in terms of Antibiotic Resistance Indices

111

(ARI) and Multiple Antibiotic Resistance Indices (MARI), respectively.

112

To the best of our knowledge, this is the first report on freshwater P. shigelloides isolates

113

from the Eastern Cape Province in South Africa, which provides a local context to the

114

antibiogram of the bacteria and indicator for assessment of EPR and ETR.

115

2.

Materials and methods

116

2.1

Study area and sampling sites

117

The study area is located within Amathole District Municipality in the Eastern Cape Province

118

(ECP) of South Africa. The district is the third populous among the six districts of the ECP. It

119

shares boundaries with the Chris Hani, Cacadu, and OR Tambo municipalities. Figure 1 show

120

the location map, some key features, and 15 sites selected on three rivers namely: Kubusie

121

river, tyhume and Kat river. The sites were selected after reconnaissance visits based on

122

presence, proximity and intensity of one or more of the following eco-socioeconomic

123

activities on the rivers’ courses such as irrigation, pastoral activities (livestock watering,

124

poultry, swine, etc.), wastewater treatment plants, recreational activities (waterfall),

125

proximity to farmland and farming activities, dam activity, domestic and household uses,

126

spiritual cleansing, water abstractions, and impoundments.

127

2.2

128

P. shigelloides were isolated from serially diluted water samples collected from the sites

129

following standard membrane filtration technique (APHA, 2005) using Inositol Brilliant

130

Green Bile Agar (details in Appendix A). The genomic DNA of the resultant presumptive P.

131

shigelloides obtained from the samples were subsequently extracted following overnight

132

cultivation on nutrient agar using the boiling method (Salerno et al., 2010) (Appendix A).

133

2.3

134

Polymerase chain reaction (PCR) for confirmation of the isolates was performed as

135

previously

136

GCAGGTTGAAGGTTGGGTAA-3′

137

which amplifies a region of the 23S rRNA gene. Four microliters of the amplification product

138

were electrophoresed in 1.5% agarose gel (Appendix A).

139

2.4

140

The antimicrobial susceptibility profiles

141

assessed using the disc diffusion method and the European Committee on Antimicrobial

142

Susceptibility Testing Guidelines (EUCAST, 2018a). A panel of 24 antibiotics including 13

143

recommended and 11 first-line antibiotic agents for the treatment of gastroenteritis and

144

extraintestinal infection were tested (details in Appendix A). All isolates were grouped as

145

Resistant (R), Intermediate (I) and Susceptible (S) to specific antibiotic based on the

146

interpretative table (CLSI, 2018; EUCAST, 2018a, 2018b).

Plesiomonas isolation and DNA extraction

Molecular confirmation of P. shigelloides

described

(Gu

and

Levin, and

2006)

using

the

primer

pair,

PS-F-5′-

PS-R-5′-TTGAACAGGAACCCTTGGTC-3′,

Antimicrobial susceptibility profiling of P. shigelloides isolates of the confirmed P. shigelloides isolates were

147

2.5

Determination of resistance quotients (RQs) of P. shigelloides isolates

148

RQs determined changes in antimicrobial resistance phenotypes of the isolates across a

149

range of antibiotics for all the sites. The RQs of the antibiotics were computed using the

150

equation (Amos et al., 2018):

151

2.6

152

assessment

153

P. shigelloides isolates from each sampling site was ‘phenotyped’ for multiple antibiotic

154

resistance ((M)ARPs) based on the 2 groups described in section 2.4. Isolates that showed

155

resistance against 3 or more antibiotics were recorded (Kinge et al., 2010). Each sampling

156

site was indexed for isolate-specific MARI (Blasco et al., 2008). The ARI was calculated for

157

each sampling site (Krumperman, 1983; Mohanta and Goel, 2014) (Appendix A).

158

The study modelled ETR and EPR based on comparative MARI and ARI values between

159

recommended and non-recommended antibiotics, respectively. The risk assessment was

160

construed on the following assumptions:

161

1. MARI (risk) based on recommended antibiotics (RAs) for treatment is always lower

162

(MARIRAs ≤ 0.2, when RAs are used) compared to non-recommended antibiotics (non-RAs,

163

MARInon-RAs ˃ 0.2). The 0.2 MARI or ARI value was an arbitrary threshold to differentiate

164

between low- and high-risk (Krumperman, 1983).

165

2. ETR = 0, when isolates are susceptible to RAs used in treatment; ETR ˃ 0, when isolates

166

are resistant against RAs. Generally, the ETR of Plesiomonas infection is defined as

167

MARIemp = MARIRAs + MARInon-RAs (where either MARIRAs or MARInon-RAs = 0, depending

168

on the group selected in an empirical treatment).

Phenotyping and indexing of isolates for multiple antibiotic resistance and risk

169

3. ARI of each site ≤ 0.2 when RAs are screened provided there is no antibiotic selection

170

pressures or pollution (ARIRAs ≤ 0.2); while ARInon-RAs ˃ 0.2 (when non-RAs are screened)

171

regardless of the presence or absence of antibiotic resistance selection pressure or pollution

172

Therefore, the EPR from a given site based on antibiotic resistance profile is defined as

173

ARIenv = ARIRAs + ARInon-RAs (where either ARIRAs or ARInon-RAs = 0, depending of group

174

selected in screening).

175

2.7

176

Hierarchical cluster analysis of antibiotic susceptibility test result was carried out by K-mean

177

and visualized using “ComplexHeatmap” r package (Gu et al., 2016) in R version 3.5.1

178

(2018-07-02). Descriptive analysis and one-way analysis of variance (ANOVA) were also

179

employed to compare different antibiotics response and sites. The difference between ARI

180

and MARI of the 2 groups of antibiotics were compared with the use of Wilcoxon signed-

181

rank test using ggpubr: 'ggplot2' R package version 0.1.8.999 (Kassambara, 2018). The

182

antibiotic resistance quotients (RQs) across the 15 sampling sites were calculated in Excel

183

version 2016. The difference among sites or antibiotics was defined as significant when P

184

values ≤ 0.05.

185

3. Results

186

Appendix B provides additional information on subsection of section 3.

187

3.1

188

A total of 182 confirmed P. shigelloides isolates were selected for antibiotic susceptibility

189

testing as follows: 35 from Kubusie river (KB1=7, KB2=3, KB3=4, KB4=14, KB5=7); 94

190

from Kat river (KT1=9, KT2=25, KT3=18, KT4=11, KT5=31); and 53 from Tyhume river

Data analysis

Confirmation of P. shigelloides

191

(TY1=8, TY2=3, TY3=9, TY4=16, TY5=17). All the 182 isolates yielded the expected

192

amplicon size (628-bp) characteristic of Plesiomonas (Figure 2).

193

3.2

194

Figure 3 presents heatmap cluster analysis of the isolates’ antibiogram fingerprints against

195

recommended antibiotics with respect to sample sites. Full graphic for both recommended

196

and non-recommended antibiotics can be found in Figure S1. The isolates generally have two

197

main clusters of antibiogram profile column-wise (Figure 3) in all the rivers, namely, a

198

cluster depicting antibiotic susceptibility pattern while the other cluster indicates antibiotic

199

resistance pattern of the isolates. For the isolates that originated from Tyhume river, 6

200

clusters characteristics of hetero-site isolates were noticed row-wise (Figure 3(A)). Figure

201

3B presents antibiogram profile clusters of isolates from Kat river. Five antibiogram clusters

202

that grouped isolates of diverse origin were obtained. For isolates from Kubusie river, 5

203

antibiogram clusters of hetero-site isolates were observed row-wise (Figure 3.C).

204

3.3

205

The occurrence of resistant isolates across sampling locations is summarised in Table S1.

206

Resistance occurence against recommended antibiotics was significantly different across

207

sampling sites (inter-sampling sites) on the rivers’ courses (Kubusie river: ANOVA, F =

208

8.675, P-value = 3.44 × 10−8; Kat river: ANOVA, F = 7.41, P-value = 3.15 × 10−7; and

209

Tyhume river: ANOVA, F = 4.336, P-value = 0.0002). Intra-site occurrence of resistant

210

isolates against different antibiotics was significantly different at Tyhume (ANOVA, F =

211

3.945, P-value = 0.01), and KAT (ANOVA, F = 3.763, P-value = 0.01) rivers’ courses, but

212

insignificantly different on Kubusie river (ANOVA, F = 1.654, P-value = 0.17). Generally,

213

high occurrence of resistance against most of the antibiotics tested compared to other sites

Antibiogram profiles and cluster analysis of isolates

Occurrence of resistant isolates per location

214

was found at KT2. Total occurrence of resistant isolates against recommended antibiotic

215

ranged from 9 (TY2/TY3) to 84 (KT2).

216

Occurrence of resistance against non-recommended antibiotics by the isolates was

217

significantly different across sites on the rivers (Tyhume river: ANOVA, F = 7.084, P-value

218

= 4.74 × 10−6; Kubusie river: ANOVA, F = 7.357, P-value = 3.11 × 10−6; and Kat river:

219

ANOVA, F = 5.639, P-value = 5.10 × 10−5). Notably, low frequency of resistance against

220

gentamycin and amikacin (0≥ n≤ 2) was observed in the isolates across locations. The

221

subtotal of resistant isolates against non-RAs across sites ranged from 16 (TY2) to 133

222

(KT5). Intra-site comparison of occurrence of resistant isolates against non-recommended

223

antibiotics showed a significantly different results in all the rivers (Tyhume river: ANOVA, F

224

= 2.670, P-value = 0.04; Kubusie river: ANOVA, F = 2.528, P-value = 0.05; and Kat river:

225

ANOVA, F = 3.451, P-value = 0.02).

226

3.4

227

The antibiotic resistance quotients (RQs) of the isolates across the 15 sampling locations is

228

shown in Table S2. All the Plesiomonas isolates had low RQS against imipenem, meropenem

229

and norfloxacin antimicrobials in all the sites with the exception of sites KT2 and KT5 for

230

imipenem and norfloxacin, respectively. Levofloxacin had 0% RQs at all sites on Kubusie

231

river, at KT4 and KT5 on Kat river, and at TY2, TY3 and TY4 on Tyhume river. The RQs of

232

the isolates against ceftriaxone, ceftazidime, trimethoprim-sulfamethoxazole, cefuroxime and

233

cefoxitin were usually high on Kubusie river at KB2, KB3, and on Kat river at KT2, and

234

KT3.

235

3.5

236

shigelloides isolates

Antibiotic resistance quotients (RQs) of Plesiomonas isolates

Descriptive

analysis

of

antibiogram

fingerprints

profiles

of

P.

237

Table 1 presents the overall descriptive analysis of antibiotic susceptibility profile of the P.

238

shigelloides isolates from the three rivers. For recommended antibiotics, percentage

239

susceptibility of the isolates ranged from 42.31% (cefuroxime) to 95.06% (imipenem). The

240

resistance of the isolates against recommended antibiotics decreased in order from cefoxitin

241

(69, 37.91%), cefuroxime (64, 35.17%), ceftazidime (62, 34.07%), cefepime (58, 31.87%),

242

ceftriaxone (54, 29.67%), ciprofloxacin (33, 18.13%), trimethoprim-sulfamethoxazole (19,

243

10.44%), piperacillin/tazobactam (16, 8.79%), ertapenem (9, 4.95%), norfloxacin (8, 4.40%),

244

levofloxacin (5, 2.75%), meropenem (2, 1.10%) and imipenem (1, 0.55%).

245

Record of isolates’ extreme resistance to some non-recommended antibiotics include

246

vancomycin (163, 89.56%), erythromycin (163, 89.56%) and sulfamethoxazole (172,

247

94.51%); and intermediate resistance to cefazolin (52, 28.57%) and amikacin (53, 29.12%).

248

About 67.58% to 80.77% of the isolates were susceptible to amikacin, gentamycin,

249

polymyxin and tetracycline.

250

3.6

251

shigelloides isolates

252

3.6.1 ETR from comparative MARI of P. shigelloides isolates

253

The comparison of MARPs and MARI of Plesiomonas isolates against recommended and

254

non-recommended antibiotics for assessment of ETR is given in Table S3. The MARI of the

255

isolates were significantly different between recommended and non-recommended antibiotics

256

(Wilcoxon, P = 12 × 10−14). While the average of the isolates MARI based on antibiotics of

257

choice was 0.168±0.181 (median/mode = 0/0.08), the mean MARI according to non-

258

recommended was 0.446±0.133 (Median/Mode = 0.45/0.45). A quick summary of Table S3

259

showed that 120 (65.93%) and 62 (34.07%) isolates had MARI of 0 − 0.15 and 0.23 − 0.62,

260

respectively for the recommended antibiotics. The MARI range of 0.09-0.18 and 0.27 − 0.82

Assessment of ETR and EPR established on MARIs and ARIs of P.

261

were noted for 3 (1.65%) and 175 (96.2%) isolates against non-recommended antibiotics.

262

Most importantly, while some isolates had zero MARI considering recommended antibiotics,

263

they had MARI ˃0.20 in case of non-recommended.

264

Summarily, the successful ETR (MARI < 0.2) and unsuccessful ETR (MARI ≥ 0.2) of P.

265

shigelloides infections due to the isolates considering recommended antibiotics collectively

266

was 65.93% and 34.07%, respectively. Similarly, the successful ETR and unsuccessful ETR

267

of infections due to the isolates on the ground of using non-recommended antibiotics was

268

1.65% and 98.35%, respectively. However, risk varied with individual isolate or antibiotics.

269

3.6.2 EPR from comparative ARI across sampling units.

270

Figure 4 shows the antibiotic resistance indices across sampling units. A single factor

271

comparison

272

antibiotics-based (nonRAs-based) ARI across the sites was significantly different (ANOVA,

273

F = 112.36, P-value = 1.18 × 10−11). The average RAs-based and nonRAs-based ARI across

274

sampling units were 0.184±0.091 and 0.459±0.050, respectively. Generally, recommended-

275

antibiotics-based ARI < 0.13 was found at KB4, KB5, KT4, KT5, TY4, TY5 and TY6; and ≥

276

0.2 at other locations. Non-RAs-based ARI had high values and ranged from 0.38 − 0.55

277

across the locations.

278

3.7

279

(M)ARPs of P. shigelloides isolates across various sampling units of the rivers against

280

recommended antibiotics (RAs) is shown in Table 2. The combinatorial expressions below

281

predicted different theoretical (M)ARPs possible for the 13 recommended and 11 non-

282

recommended antibiotics (nonRAs) as 8192 and 2048, respectively:

283

n

Cr =

of

recommended-antibiotics-based

(RAs-based) and

non-recommended-

Multi-resistance patterns ((M)ARPs) of P. shigelloides isolates

for RAs (n = 13, r = 0, 1 … 13) and;

284

n

285

However, only 61 and 68 (M)ARPs against RAs and nonRAs (data not shown) were

286

observed. Of the 182 isolates, 63 (34.62%) were sensitive to RAs, while 36 (19.8%) showed

287

resistance to 1 antibiotic. Also, 6.04% (n = 11) to 28.6% (n = 52) of the isolates had 3 to ≥5

288

(M)ARPs. For distribution of the observed 61 (M)ARPs across sampling locations, cefoxitin

289

had highest resistance proportion (9.29%). Four (2.19%) isolates were resistant to ceftazidime

290

and ciprofloxacin each. Occasional (M)ARP (resistant found only in one isolate) was seen in

291

16.48% (n = 30) of the isolates. The most frequent (M)ARPs was cefuroxime/cefoxitin

292

observed

293

ceftriaxone/ceftazidime/cefepime/cefuroxime/cefoxitin

294

ceftriaxone/ceftazidime/ciprofloxacin/cefepime/cefuroxime/cefoxitin (5, 2.74% isolates), and

295

ceftriaxone/ceftazidime/trimethoprim-sulfamethoxazole/

296

piperacillin/tazobactam/cefepime/cefuroxime/cefoxitin (5, 2.74% isolates).

297

Different (M)ARPs were observed among the Plesiomonas isolates from 15 locations.

298

Isolates from the sites exhibited ≥ 3 (M)ARPs. Notably, the study observed a high number of

299

(M)ARPs at sites KT5 (17), followed by KT3(13), KT2(12), and TY5/6(10). The number of

300

(M)ARPs at KB5, TY2, TY3, KB3, TY4, KB1, KB2, KB4, KT4, TY1, and KT1(8) ranged

301

from 3 – 8.

302

3.0

Discussion

303

3.1.

Antimicrobial resistance and sample sites relationship

304

Antibiotic therapy remains a major practice in infection control and treatment. Here, we

305

performed antibiogram fingerprint of P. shigelloides isolates against 24 panels of antibiotics.

306

The detection and confirmation of P. shigelloides from the sampling sites can be attributed to

307

possible anthropogenic pollution along the riverbanks. All the sites serve as animal watering

Cr =

for nonRAs (n = 11, r = 0, 1 … 11).

in

10

isolates

(5.47%), (8,

followed 4.4%

by isolates),

308

point in addition to other purposes except TY1, which is chiefly hotspot for

309

swimming/recreational activities. The rivers receive wastewater effluents (WWE) upstream at

310

KT1 and KB5. All these activities could contribute substances that is capable of directly or

311

indirectly induce resistance in microorganisms. For instance, WWE are usually laden with

312

xenogenetic/xenobiotic compound that are capable of inducing resistance in the pathogen.

313

Cluster analysis of the isolates’ antibiogram fingerprints showed diversity in terms of intra-

314

sampling and inter-sampling location, which suggests that (M)ARPs are not site specific. It is

315

not impossible that the clustered isolates have the same origin. Livestock can shed clones of

316

resistant bacteria when they visit sites that are very close to one another such as KB2 and

317

KB3. Heatmap/hierarchical cluster techniques have been used to infer antimicrobial

318

susceptibility profile similarity and antibiotic disturbance on aquatic microbial composition in

319

recent times (McCusker et al., 2019).

320

Despite the antibiogram profile similarity, occurrence of resistant isolates varied significantly

321

with sampling sites. This variation suggests different intensities of anthropogenic pollutions

322

at the sites. The differences in resistance pattern to different classes of antibiotics from the

323

sites could also be attributed to presence of different type of pollutants in the sites. For

324

instance, occurrence of resistant isolates was obvious at sites in wastewater treatment plant

325

(KT1) because wastewater is commonly laden with vast arrays of antibiotic wastes and heavy

326

metals; human settlements (KB3, KB4, KT2, TY3, TY4, TY5/6) which could connote the

327

presence of different antibiotic-resistance-inducing substances in domestic wastes such as

328

disinfectants from various settlements; game farm (KB1) and livestock farm (swine, poultry,

329

sheep and goats e.g. KB1, KB2 and KB5) and different farm inputs in orchids proximities on

330

the rivers’ routes. Extended-spectrum beta-lactamase producing E. coli and fluoroquinolone-

331

resistant isolates have been reported in free-living deer and wild boars in Vojvodina Province,

332

Serbia (Velhner et al. 2018). Wastewater treatment plant effluents (WTPEs) might be

333

responsible for burden of P. shigelloides resistance at KT1. WTPEs are known to contribute

334

ARGs and antimicrobial resistant bacteria to receiving waterbodies ( Hembach et al., 2017;

335

Antti et al., 2018; Sabri et al., 2018). A high relative abundance of antimicrobial resistance

336

genes (ARGs) are common in agriculture impacted watersheds (Zhu et al. 2018). Various

337

farm and livestock management practices in the rivers’ neighbourhoods serve as point and

338

diffused sources of pesticides, herbicides, disinfectants, residual antibiotics and antibiotic

339

metabolic by-products, quaternary ammonium compounds, heavy metals and biocides, which

340

could have promoted P. shigelloides resistance in the receiving river. These substances are

341

known to select microorganisms for ARGs and/or promote horizontal gene transfer among

342

microbial communities.

343

susceptibility of S. enterica and E. coli to multiple antibiotics due to commercial herbicides

344

such as dicamba, glyphosate, 2,4-D and their co-formulants. Also, sub-inhibitory

345

concentration of biocides (triclosan and chlorhexidine), and antibiotics (sulfamethoxazole and

346

gentamicin) have been reported to significantly increased occurrences of ARGs dissemination

347

(Jutkina et al. 2018). Zhao et al. (2017) found that heavy metals (Cr; Cu, Hg, Pb, Zn) and

348

nutrients from mariculture significantly co-driven propagation of macrolide-lincosamide-

349

streptogramin B, fluoroquinolone, aminoglycoside, beta-lactam and tetracycline resistance

350

genes in adjacent environment.

351

Aside from high possibility of diffused and run-off dissemination of the afore-mentioned

352

substances and resistant bacteria selected by their pressures from these farms, direct faecal

353

shedding of resistant P. shigelloides isolates by free ranging animals or livestock could also

354

add to the observed resistance burden in P. shigelloides isolates. Litters of animal faeces were

355

seen at the locations during sampling. Furthermore, human excreta and babies pampers

356

among many other human generated wastes were noticed at KB4. This is indicative that some

357

of the resistant P. shigelloides isolates might originated from human faeces. This is similar to

Kurenbach et al. (2017) found changes in the antibiotic

358

observation of Adesiyan et al. (2019) who found high resistant of P. shigelloides isolates

359

against important antibiotics in rivers impacted with human excreta and washing activities in

360

South-western Nigeria. The probable use of animal manure and poultry dugs in farms along

361

the riverbanks may serve as another source of resistant P. shigelloides isolates or could

362

promote transfer of resistance gene to P. shigelloides in the adjoining riverbanks. Jia et al.

363

(2017) and Fang et al. (2018) found high migration of chloramphenicol, beta-lactam,

364

quinolone, trimethoprim, fosmidomycin, macrolide-lincosamide-streptogramin, polymyxin,

365

vancomycin,

366

aminoglycoside, acridine, fluoroquinolone and multidrug resistance genes from pig farm to

367

adjoining rivers in China. Also, Pornsukarom and Thakur (2017) reported horizontal transfer

368

of beta-lactam resistance genes, tetracycline resistance genes, sulphonamide, aminoglycoside

369

resistance and AMR determinants in Salmonella serotypes after land application of manure

370

from commercial swine farms.

371

The high occurrence of P. shigelloides resistant isolates against non-recommended antibiotics

372

depicts intrinsic resistance of the microorganism against the antibiotics. Although,

373

Plesiomonas isolates were susceptible to gentamycin and amikacin across the sampling

374

locations, this however, does not guarantee an in vivo performance of the antibiotics as these

375

agents are not targets for therapy (EUCAST, 2018b).

376

3.2.

377

The observed low RQs (0%) of imipenem, meropenem, norfloxacin and levofloxacin in most

378

of the sites suggests lack of antibiotic resistance selection pressures for the antibiotics in the

379

sites. Other recommended antibiotics with higher RQs imply isolates’ response to probable

380

presence of antibiotic selection pressures at the sites. High RQs have significantly associated

381

with sites heavily impacted by WTPEs and antibiotics pollution (Amos et al. 2018).

glycopeptide,

sulphonamide,

lincosamide,

macrolide,

tetracycline,

Antibiotic resistance quotients (RQs) and antibiotic susceptibility profiles

382

The resistance shown against antibiotics tested by Plesiomonas in this study is in agreement

383

with literature. Many authors have reported clinical Plesiomonas isolates’ (multi)resistance

384

against many of the tested antibiotics (Depaola et al., 1995; Auxiliadora-Martins et al.,

385

2010a;Jun et al., 2011; Bonatti et al., 2012; Chen et al., 2013; Abdelhamed et al., 2018).

386

Similar to the observed resistance against fluoroquinone, carbapenems and cephalosporins in

387

this study, Adesiyan et al. (2019) reported resistance to trimethoprim + sulphamethoxazole,

388

ciprofloxacin, norfloxacin, and imipenem in 36%, 30%, 30%, and 18% of environmental

389

isolates of P. shigelloides, respectively. High susceptibility of environmental P. shigelloides

390

isolates

391

piperacillin/tazobactam, cefepime, meropenem, cefoxitin, norfloxacin, imipenem, ertapenem,

392

gentamicin and tetracycline is in agreement with other authors (Auxiliadora-Martins et al.,

393

2010a; Matsuyama et al., 2015; Xia et al., 2015).

394

The MARI of P. shigelloides isolates established on recommended and non-recommended

395

antibiotics was significantly different and attests to the inherent potency of the two groups of

396

antibiotics. With modal value of the isolates’ MARI equals zero for recommended drugs

397

while the corresponding value for non-recommended antibiotics was 0.45, it implies an

398

underlying risk that should normally be associated with empirical treatment of P. shigelloides

399

infections with the antibiotic groups if there is no resistance case. The estimated EPR of P.

400

shigelloides infection in this study suggests that, should empirical treatment of infection be

401

necessary in the communities, clinicians that opt for any of the recommended antibiotics

402

against the usual first line agents (non-recommended) prior to proper diagnosis have 65.93%

403

chance of avoiding fatal scenario or outcome. However, early diagnosis of P. shigelloides

404

extraintestinal infections as well as the use of appropriate antibiotics treatment have been

405

advised (Xia et al., 2015). Late diagnosis, misdiagnosis and wrong antibiotics treatment lead

406

to high mortality rate (Xia et al., 2015). Several cases of empirical treatment and late

to

trimethoprim-sulfamethoxazole,

levofloxacin,

ciprofloxacin,

407

diagnosis of P. shigelloides infection have been reported to cause death in extraintestinal

408

infections (Billiet et al., 1989; Nolte et al., 1988; Terpeluk et al., 1992). Some of the non-

409

recommended antibiotics reported in unsuccessful empirical (combinatorial) treatment of P.

410

shigelloides extraintestinal infections include ampicillin, gentamicin and vancomycin ( Nolte

411

et al., 1988;Billiet et al., 1989; Terpeluk et al., 1992). Empirical treatments that involved at

412

least one of the appropriate antibiotics as first-line agent increased survival rate (Waecker et

413

al., 1988; Fujita et al., 1994).

414

ARI established by recommended antibiotics proved a good indicator for assessment of EPR

415

compared to non-recommended antibiotics as the group modal and mean values were both

416

lower than 0.2. Meanwhile, non-recommended antibiotics had group modal and mean ARI

417

values greater than 0.2 even in the absence of potential environmental pollution or resistance

418

selection pressures. The ARI values greater than 0.2 for recommended antibiotics at KB1,

419

KT1, TY2, KB2, KB3, KT2, KT3 and TY3 identified possible environmental pollution.

420

However, ARI values lower than 0.2 at TY5, KB4 and KT5, where a number of (M)ARPs

421

were observed is indicative of direct inputs from point/diffuse source pollutions rather than

422

presence of selection pressure. Sewage contamination could reintroduced resistant P.

423

shigelloides into natural habitats (Foster et al., 2000).

424

The use of comparative approach of ARI/MARI between recommended antibiotics and non-

425

recommended antibiotics against P. shigelloides further alerts the need for caution whenever

426

Krumperman’s MARI value of 0.2 threshold reference (Krumperman, 1983) is been applied

427

for delimiting low- and high-risk contamination. Because antimicrobials to which a microbe

428

is intrinsically resistance against produce MARI/ARI > 0.2. While several isolates in this

429

study had MARI value of 0 against recommended antibiotics, the same had values >0.2

430

against non-recommended antibiotics (Table S3).

431

Multiple resistance of P. shigelloides to important antibiotics could have serious clinical

432

consequences. The observed differential sensitivity or resistance of the isolates to antibiotics

433

from the same antimicrobial class could be linked to acquisition of different copies of ARG

434

or accumulation of point mutations. For instance, carbapenem resistance in Gram-negative

435

bacteria involve expression of efflux pumps, beta-lactamases, porin loss/mutation and

436

alterations in penicillin binding proteins (Papp-Wallace et al., 2011; Meletis, 2016).

437

Quinolones and fluoroquinolones resistance involve mutations in DNA gyrase and

438

topoisomerase IV, plasmid-borne resistance genes such as Qnr proteins and Aac(6′)-Ib-cr

439

aminoglycoside acetyltransferase, efflux pumps, reduced expression of porins and

440

overexpression of chromosome-encoded efflux pumps (Aldred et al., 2014). One isolate

441

might acquire multiple of these mechanisms, which then accounts for its varied resistance to

442

members of the same antimicrobial class.

443

4. Conclusion

444

The findings revealed an unusual P. shigelloides resistance against cephalosporins,

445

quinolones and fluoroquinolones. The resistance observed in the isolates is reflective of the

446

qualities of the freshwaters and consequence of anthropogenic activities in the catchments.

447

MARI attributed low risk of ET to recommended panel of antibiotics but higher risk to non-

448

recommended antibiotics. The likelihood of successful ET of P. shigelloides infections due to

449

resistance in the isolates using recommended antibiotics was very higher compared to low

450

chance of success obtained in the case of non-recommended antibiotics. ARI based on

451

recommended antibiotics identified potential likelihood of environmental pollutions at KB2,

452

KB3, KT2, KT3 and TY3. Resistance in P. shigelloides especially against cephalosporin,

453

quinolones and fluoroquinolones is distressing and suggests high pollution loading of the

454

freshwaters with substances with potential public health concerns in the Eastern Cape

455

Province.

Future

research

should

consider

direct

quantification

456

antimicrobials/antimicrobial metabolic by-product, pesticides, herbicides and other

457

xenobiotics/xenogenetic elements in the freshwater and their relevant hazard quotients in the

458

catchment.

459

Acknowledgements

460

The authors thank the South Africa Medical Research Council (SAMRC) and the National

461

Research Foundation, The World Academy of Science (NRF-TWAS) for financial support

462

(Grant Numbers: 99796 and 116382). Conclusions arrived at and opinions expressed in this

463

article are those of the authors and are not necessarily to be attributed to SAMRC or NRF-

464

TWAS.

465

Supplementary data

466

Appendix A

467

Appendix B

468

Tables S1 to S3

469

Figure S1

470

References

471

Abdelhamed, H., Ozdemir, O., Tekedar, H.C., Arick, M.A., Hsu, C.-Y., Karsi, A., Lawrence,

472

M.L., 2018. Complete genome sequence of multidrug-resistant Plesiomonas shigelloides

473

strain MS-17-188. Genome Announc., 6, pp.e00387-18

474

https://doi.org/10.1128/genomeA.00387-18

475

Ahmed, W., Zhang, Q., Lobos, A., Senkbeil, J., Sadowsky, M.J., Harwood, V.J., Saeidi, N.,

476

Marinoni, O., Ishii, S., 2018. Corrigendum to “Precipitation influences pathogenic

of

477

bacteria and antibiotic resistance genes abundance in storm drain outfalls in coastal sub-

478

tropical waters” [Environ. Int. 116, 308–

479

318](S0160412018301685)(10.1016/j.envint.2018.04.005). Environ. Int. 119, 239.

480

https://doi.org/10.1016/j.envint.2018.06.031

481

Alcaniz, J.P., de Cuenca Moron, B., Rubio, M.G., Albares, J.L.M., Alvarez, J.G., 1995.

482

Spontaneous bacterial peritonitis due to Plesiomonas shigelloides. Am. J. Gastroenterol.,

483

90, 1529-1530.

484 485 486 487 488

Aldová, E., Melter, O., Chýle, P., Šlosárek, M., Kodym, P., 1999. Plesiomonas shigelloides in water and fish. Cent. Eur. J. Public Health,7, 172-175. PMID:10659376 Aldred, K.J., Kerns, R.J., Osheroff, N., 2014. Mechanism of quinolone action and resistance. Biochemistry, 53(10), 1565-1574. https://doi.org/10.1021/bi5000564 Alexander, S., Fazal, M.-A., Burnett, E., Deheer-Graham, A., Oliver, K., Holroyd, N.,

489

Parkhill, J., Russell, J.E., 2016. Complete genome sequence of Plesiomonas shigelloides

490

Type Strain NCTC10360. Genome Announc., 4, e01031-16.

491

https://doi.org/10.1128/genomeA.01031-16

492

Amos, G.C.A., Ploumakis, S., Zhang, L., Hawkey, P.M., Gaze, W.H., Wellington, E.M.H.,

493

2018. The widespread dissemination of integrons throughout bacterial communities in a

494

riverine system. ISME J. 12, 681–691. https://doi.org/10.1038/s41396-017-0030-8

495

Ampofo, K., Graham, P., Ratner, A., Rajagopalan, L., Della-Latta, P., Saiman, L., 2001.

496

Plesiomonas shigelloides sepsis and splenic abscess in an adolescent with sickle-cell

497

disease. Pediatr. Infect. Dis. J. 20, 1178–1179. https://doi.org/10.1097/00006454-

498

200112000-00019

499

Antti, K., Katariina, P., Joakim, L.D.G., 2018. Fecal pollution explains antibiotic resistance

500

gene abundances in anthropogenically impacted environments. BioRxiv, 341487.

501

https://doi.org/10.1101/341487

502

APHA, 2005. Standard Methods for the Examination of Water and Wastewater, American

503

Water Works Association/American Public Works Association/Water Environment

504

Federation. https://doi.org/10.2105/AJPH.51.6.940-a

505

Arai, T., Ikejima, N., Itoh, T., Sakai, S., Shimada, T., Sakazaki, R., 1980. A survey of

506

Plesiomonas shigelloides from aquatic environments, domestic animals, pets and

507

humans. J. Hyg. (Lond). 84, 203–11. https://doi.org/10.1017/S002217240002670X

508

Auxiliadora-Martins, M., Bellissimo-Rodrigues, F., Viana, J.M., Alkmin Teixeira, G.C.,

509

Nicolini, E.A., Muniz Cordeiro, K.S., Colozza, G., Martinez, R., Martins-Filho, O.A.,

510

Basile-Filho, A., Teixeira, G.C.A., Nicolini, E.A.., Cordeiro, K.S., Colozza, G.,

511

Martinez, R., Martins-Filho, O.A., Basile-Filho, A., Alkmin Teixeira, G.C., Nicolini,

512

E.A., Muniz Cordeiro, K.S., Colozza, G., Martinez, R., Martins-Filho, O.A., Basile-

513

Filho, A., 2010a. Septic shock caused by Plesiomonas shigelloides in a patient with

514

sickle beta-zero thalassemia. Hear. LUNG 39, 335–339.

515

https://doi.org/10.1016/j.hrtlng.2009.06.015

516

Auxiliadora-Martins, M., Bellissimo-Rodrigues, F., Viana, J.M., Teixeira, G.C.A., Nicolini,

517

E.A., Cordeiro, K.S.M., Colozza, G., Martinez, R., Martins-Filho, O.A., Basile-Filho,

518

A., 2010b. Septic shock caused by Plesiomonas shigelloides in a patient with sickle

519

beta-zero thalassemia. Hear. Lung J. Acute Crit. Care 39, 335–339.

520

https://doi.org/10.1016/j.hrtlng.2009.06.015

521

Avison, M.B., Bennett, P.M., Walsh, T.R., 2000. beta-lactamase expression in Plesiomonas

522

shigelloides. J. Antimicrob. Chemother. 45, 877–880.

523

https://doi.org/10.1093/jac/45.6.877

524 525 526

Bardon, J., 1999. Evaluation of the pathogenicity of strains of Plesiomonas shigelloides isolated in animals. Vet. Med. (Praha). 44, 161–164. Billiet, J., Kuypers, S., Van Lierde, S., Verhaegen, J., 1989. Plesiomonas shigelloides

527

meningitis and septicaemia in a neonate: report of a case and review of the literature. J.

528

Infect. Journal of Infection, 19, 267-271. https://doi.org/10.1016/S0163-4453(89)90809-

529

8

530

Blasco, M.D., Esteve, C., Alcaide, E., 2008. Multiresistant waterborne pathogens isolated

531

from water reservoirs and cooling systems. J. Appl. Microbiol., 105, 469-475.

532

https://doi.org/10.1111/j.1365-2672.2008.03765.x

533

Bonatti, H., Sifri, C., Sawyer, R.G., 2012. Successful liver transplantation from donor with

534

Plesiomonas shigelloides sepsis after freshwater drowning: case report and review of

535

literature on Gram-Negative bacterial aspiration during drowning and utilization of

536

organs from bacteremic donors. Surg. Infect. (Larchmt). 13, 114–120.

537

https://doi.org/10.1089/sur.2010.018

538

Bowman, J.K., Zhang, X.C., Hack, J.B., 2016. Plesiomonas shigelloides meningitis in an

539

adult in the ED, The American Journal of Emergency Medicine, 34:1329e1–1329e2.

540

https://doi.org/10.1016/j.ajem.2015.12.046

541

Brenner, D., Krieg, N.R., Staley, J.T., Garrity, G.M., 2005. Bergey’s Manual® of Systematic

542

Bacteriology. Bergey’s Manual® Syst. Bacteriol. 333–379. https://doi.org/10.1007/0-

543

387-29298-5

544

Calero-Cáceres, W., Muniesa, M., 2016. Persistence of naturally occurring antibiotic

545

resistance genes in the bacteria and bacteriophage fractions of wastewater. Water Res.

546

95, 11–18. https://doi.org/10.1016/j.watres.2016.03.006

547

Canosa, A., Pinilla, G., 1999. Bacteriological eutrophication indicators in four Colombian

548

water bodies (South America). Lakes Reserv. Res., 4, 23-27.

549

https://doi.org/10.1046/j.1440-1770.1999.00079.x

550

Chen, Q., An, X., Li, H., Su, J., Ma, Y., Zhu, Y.G., 2016. Long-term field application of

551

sewage sludge increases the abundance of antibiotic resistance genes in soil. Environ.

552

Int. 92–93, 1–10. https://doi.org/10.1016/j.envint.2016.03.026

553

Chen, X., Chen, Y., Yang, Q., Kong, H., Yu, F., Han, D., Zheng, S., Cui, D., Li, L., 2013.

554

Plesiomonas shigelloides Infection in Southeast China. PLoS One 8.

555

https://doi.org/10.1371/journal.pone.0077877

556

Claesson, B.E., Holmlund, D.E., Lindhagen, C.A., Mätzsch, T.W., 1984. Plesiomonas

557

shigelloides in acute cholecystitis: a case report. J. Clin. Microbiol. 20, 985–987. 20,

558

985–7. https://jcm.asm.org/content/jcm/20/5/985.full.pdf

559

Clark, R.B., Randolph Westby, G., Spector, H., Soricelli, R.R., Young, C.L., 1991. Fatal

560

Plesiomonas shigelloides septicaemia in a splenectomised patient. J. Infect., 23, 89-92.

561

https://doi.org/10.1016/0163-4453(91)94217-8

562 563 564

CLSI, 2018. Performance Standards for Antimicrobial Susceptibility Testing, in: CLSI Supplement M100. Dahm, L.J., Weinberg, A.G., 1980. Plesiomonas (Aeromonas) shigelloides septicemia and

565

meningitis in a neonate. South. Med. J. 73, 393–394. https://doi.org/10.1097/00007611-

566

198003000-00039

567

Depaola, A., Peeler, J.T., Rodrick, G.E., 1995. Effect of oxytetracycline-medicated feed on

568

antibiotic-resistance of gram-negative bacteria in catfish ponds. Appl. Environ.

569

Microbiol. 61, 2335–2340. https://aem.asm.org/content/aem/61/6/2335.full.pdf

570

Ding, X., Peng, X.J., Jin, B.S., Xiao, M., Chen, J.K., Li, B., Fang, C.M., Nie, M., 2015.

571

Spatial distribution of bacterial communities driven by multiple environmental factors in

572

a beach wetland of the largest freshwater lake in China. Front. Microbiol. 6, 1–9.

573

https://doi.org/10.3389/fmicb.2015.00129

574 575

Dudley, A.G., Mays, W., Sale, L., 1982. Plesiomonas (Aeromonas) shigelloides meningitis in a neonate--a case report. J. Med. Assoc. Ga. 71, 775–776. PMID:7175386

576

Eason, J.D., Peacock, D., 1996. Plesiomonas shigelloides septicemia and meningitis in a

577

neonate. Can. J. Infect. Dis., 7, 380-382. https://doi.org/10.1155/1996/404780

578

EUCAST, 2018a. Plesiomonas shigelloides calibration of zone diameter breakpoints to MIC

579

values Plesiomonas shigelloides MIC and zone diameter correlates.

580

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_criteria/Validatio

581

n_2018/Plesiomonas_shigelloides_v_1.0_June_2018.pdf

582

EUCAST, 2018b. Enterobacteriaceae (Enterobacterales). Enterobact. Calibration Zo. Diam.

583

Break. to MIC values.

584

http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_criteria/Validatio

585

n_2018/Enterobacteriaceae_v_6.0_June_2018.pdf

586

Fang, H., Han, L., Zhang, H., Long, Z., Cai, L., Yu, Y., 2018. Dissemination of antibiotic

587

resistance genes and human pathogenic bacteria from a pig feedlot to the surrounding

588

stream and agricultural soils. J. Hazard. Mater. 357, 53–62.

589

https://doi.org/10.1016/j.jhazmat.2018.05.066

590

Fischer, K., Chakraborty, T., Hof, H., Kirchner, T., Wamsler, O., 1988. Pseudoappendicitis

591

caused by Plesiomonas shigelloides. J. Clin. Microbiol. 26, 2675–2677.

592

https://jcm.asm.org/content/jcm/26/12/2675.full.pdf

593

Foster, G., Patterson, T., Pennycott, T., Cheasty, T., Jagger, T., Keane, S., Robertson, S.,

594

2000. Plesiomonas shigelloides - an uncommon cause of diarrhoea in cats? Vet. Rec.

595

146, 296.

596

Fujita, K., Shirai, M., Ishioka, T., Kakuya, F., 1994. Neonatal Plesiomonas shigelloides

597

septicemia and meningitis: A case and review. Pediatr. Int., 36, 450-452.

598

https://doi.org/10.1111/j.1442-200X.1994.tb03222.x

599

Gibotti, A., Saridakis, H.O., Pelayo, J.S., Tagliari, K.C., Falcão, D.P., 2000. Prevalence and

600

virulence properties of Vibrio cholorae non-O1 Aeromonas spp. and Plesiomonas

601

shigelloides isolated from Cambe Stream (State of Parana, Brazil). J. Appl. Microbiol.,

602

89, 70-75. https://doi.org/10.1046/j.1365-2672.2000.01077.x

603

Gonzalez-Rey, C., Svenson, S.B., Eriksson, L.M., Ciznar, I., Krovacek, K., 2003.

604

Unexpected finding of the “tropical” bacterial pathogen Plesiomonas shigelloides from

605

lake water north of the Polar Circle. Polar Biol. 26, 495–499.

606

https://doi.org/10.1007/s00300-003-0521-0

607

Gopal, V., Burns, F.E., 1991. Cellulitis and compartment syndrome due to Plesiomonas

608

shigelloides: a case report. Mil. Med., 156, 43-43.

609

https://doi.org/10.1093/milmed/156.1.43

610

Graciaa, D.S., Cope, J.R., Roberts, V.A., Cikesh, B.L., Kahler, A.M., Vigar, M., Hilborn,

611

E.D., Wade, T.J., Backer, L.C., Montgomery, S.P., Evan Secor, W., Hill, V.R., Beach,

612

M.J., Fullerton, K.E., Yoder, J.S., Hlavsa, M.C., 2018. outbreaks associated with

613

untreated recreational water — United States, 2000–2014. Am. J. Transplant. 18, 2083–

614

2087. https://doi.org/10.1111/ajt.15002

615 616

Gu, W.M., Levin, R.E., 2006. Factors affecting quantitative PCR assay of Plesiomonas shigelloides. Food Biotechnol. 20, 219–230.

617 618

https://doi.org/10.1080/08905430600709750 Gu, Z., Eils, R., Schlesner, M., 2016. Complex heatmaps reveal patterns and correlations in

619

multidimensional genomic data. Bioinformatics, 32, 2847-2849.

620

https://doi.org/10.1093/bioinformatics/btw313

621

Guo, X. pan, Yang, Y., Lu, D. pei, Niu, Z. shun, Feng, J. nan, Chen, Y. ru, Tou, F. yun,

622

Garner, E., Xu, J., Liu, M., Hochella, M.F., 2018. Biofilms as a sink for antibiotic

623

resistance genes (ARGs) in the Yangtze Estuary. Water Res. 129, 277–286.

624

https://doi.org/10.1016/j.watres.2017.11.029

625

Harnisz, M., Korzeniewska, E., 2018. The prevalence of multidrug-resistant Aeromonas spp.

626

in the municipal wastewater system and their dissemination in the environment. Sci.

627

Total Environ. 626, 377–383. https://doi.org/10.1016/j.scitotenv.2018.01.100

628

Hembach, N., Schmid, F., Alexander, J., Hiller, C., Rogall, E.T., Schwartz, T., 2017.

629

Occurrence of the mcr-1 colistin resistance gene and other clinically relevant antibiotic

630

resistance genes in microbial populations at different municipal wastewater treatment

631

plants in Germany. Front. Microbiol. 8, 1–11. https://doi.org/10.3389/fmicb.2017.01282

632

Holmberg, S.D., Farmer, J.J., 1984. Aeromonas hydrophila and Plesiomonas shigelloides as

633

causes of intestinal infections. Rev. Infect. Dis., 6, 633-639.

634

https://doi.org/10.1016/S0196-4399(85)80037-4

635

Holmberg, S.D., Wachsmuth, I.K., Hickman-Brenner, F.W., Blake, P.A., Farmer, J.J., 1986.

636

Plesiomonas enteric infections in the United States. Ann. Intern. Med. 105, 690–694.

637

https://doi.org/10.7326/0003-4819-105-5-690

638

Huber, I., Spanggaard, B., Appel, K.F., Rossen, L., Nielsen, T., Gram, L., 2004. Phylogenetic

639

analysis and in situ identification of the intestinal microbial community of rainbow trout

640

(Oncorhynchus mykiss, Walbaum). J Applied Microbiol., 96, 117-132.

641

https://doi.org/10.1046/j.1365-2672.2003.02109.x

642

Hustedt, J.W., Ahmed, S., 2017a. Plesiomonas shigelloides Periprosthetic Knee Infection

643

After Consumption of Raw Oysters. Am. J. Orthop. (Belle Mead. NJ), 46(1), E32-E34.

644

PMID:28235124

645

Hustedt, J.W., Ahmed, S., 2017b. Infection After Consumption of Raw Oysters 32–34.

646

Islam, M.S.L., Alam, J.R., Khan, S.I., 1991. Distribution of Plesiomonas shigelloides in

647

Various Components of Pond Ecosystems in Dhaka, Bangladesh. Microbiol. Immunol.,

648

35, 927-932. https://doi.org/10.1111/j.1348-0421.1991.tb01614.x

649

Jiang, H., Zhou, R., Yang, Y., Chen, B., Cheng, Z., Zhang, M., Li, J., Zhang, G., Zou, S.,

650

2018. Characterizing the antibiotic resistance genes in a river catchment: Influence of

651

anthropogenic activities. J. Environ. Sci. (China) 69, 125–132.

652

https://doi.org/10.1016/j.jes.2017.08.009

653

Jiao, Y.N., Chen, H., Gao, R.X., Zhu, Y.G., Rensing, C., 2017. Organic compounds stimulate

654

horizontal transfer of antibiotic resistance genes in mixed wastewater treatment systems.

655

Chemosphere 184, 53–61. https://doi.org/10.1016/j.chemosphere.2017.05.149

656

Jun, J.W., Kim, J.H., Choresca Jr., C.H., Shin, S.P., Han, J.E., Jeong, D.S., Park, S.C., 2011.

657

Isolation and molecular detection of Plesiomonas shigelloides containing tetA gene from

658

Asian arowana (Scleropages formosus) in a Korean aquarium. African J. Microbiol. Res.

659

5, 5019–5021. https://doi.org/10.5897/AJMR11.678

660

Kain, K.C., Kelly, M.T., 1989. Antimicrobial susceptibility of Plesiomonas shigelloides from

661

patients with diarrhea. Antimicrob. Agents Chemother., 33, 1609-1610.

662

https://doi.org/10.1128/AAC.33.9.1609

663

Kain, K.C., Kelly, M.T., 1989a. Clinical features, epidemiology, and treatment of

664

Plesiomonas shigelloides diarrhea. J. Clin. Microbiol., 27, 998-1001.

665

https://jcm.asm.org/content/jcm/27/5/998.full.pdf

666

Kain, K.C., Kelly, M.T., 1989b. Antimicrobial susceptibility of Plesiomonas shigelloides

667

from patients with diarrhea. Antimicrob. Agents Chemother., 33, 1609-1610.

668

https://doi.org/10.1128/AAC.33.9.1609

669 670 671

Karkman, A., Do, T.T., Walsh, F., Virta, M.P.J., 2017. Antibiotic-Resistance Genes in Waste Water. Trends Microbiol. 26, 220–228. https://doi.org/10.1016/j.tim.2017.09.005 Kinge, C.N.W., Ateba, C.N., Kawadza, D.T., 2010. Antibiotic resistance profiles of

672

Escherichia coli isolated from different water sources in the mmabatho locality, North-

673

West Province, South Africa. S. Afr. J. Sci. 106, 44–49.

674

https://doi.org/10.4102/sajs.v106i1/2.14

675

Klatte, J.M., Dastjerdi, M.H., Clark, K., Harrison, C.J., Grigorian, F., Stahl, E.D., 2012.

676

Hyperacute infectious keratitis with Plesiomonas shigelloides following traumatic

677

lamellar corneal laceration. Pediatr Infect Dis J 31, 1200–1201.

678

https://doi.org/10.1097/INF.0b013e318266b61f

679

Klontz, E.H., Faruque, A.S.G., Das, S.K., Malek, M. a., Islam, Z., Luby, S.P., Klontz, K.C.,

680

2012. Clinical and Epidemiologic Features of Diarrheal Disease due to Aeromonas

681

hydrophila and Plesiomonas shigelloides Infections Compared with Those due to Vibrio

682

cholerae Non-O1 and Vibrio parahaemolyticus in Bangladesh. ISRN Microbiol. 2012,

683

1–6. https://doi.org/10.5402/2012/654819

684

Korzeniewska, E., Korzeniewska, A., Harnisz, M., 2013. Antibiotic resistant Escherichia coli

685

in hospital and municipal sewage and their emission to the environment. Ecotoxicol.

686

Environ. Saf., 91, 96-102. https://doi.org/10.1016/j.ecoenv.2013.01.014

687

Krovacek, K., Eriksson, L.M., González-Rey, C., Rosinsky, J., Ciznar, I., 2000. Isolation,

688

biochemical and serological characterisation of Plesiomonas shigelloides from

689

freshwater in Northern Europe. Comp. Immunol. Microbiol. Infect. Dis. 23, 45–51.

690

https://doi.org/10.1016/S0147-9571(99)00058-2

691

Krumperman, P.H., 1983. Multiple antibiotic resistance indexing of Escherichia coli to

692

identify high-risk sources of fecal contamination of foods . Multiple Antibiotic

693

Resistance Indexing of Escherichia coli to Identify High-Risk Sources of Fecal

694

Contamination of Foodst. Appl. Environ. Microbiol. 46, 165–170.

695

https://doi.org/10.1007/s11356-014-3887-3

696

Kwaga, J.K., Adesiyun, A.A., Bello, C.S., Abdullahi, S.U., 1988. Occurrence of Plesiomonas

697

shigelloides in humans and water in Zaria, Nigeria. Microbiologica, 11, 165-167.

698

PMID:3405099

699

Leonard, A.F.C.C., Zhang, L., Balfour, A.J., Garside, R., Gaze, W.H., 2015. Human

700

recreational exposure to antibiotic resistant bacteria in coastal bathing waters. Environ.

701

Int. 82, 92–100. https://doi.org/10.1016/j.envint.2015.02.013

702

Li, N., Zhang, X., Wu, W., Zhao, X., 2014. Occurrence, seasonal variation and risk

703

assessment of antibiotics in the reservoirs in North China. Chemosphere 111, 327–335.

704

https://doi.org/10.1016/j.chemosphere.2014.03.129

705

Li, S., Shi, W., Li, H., Xu, N., Zhang, R., Chen, X., Sun, W., Wen, D., He, S., Pan, J., He, Z.,

706

Fan, Y., 2018. Antibiotics in water and sediments of rivers and coastal area of Zhuhai

707

City, Pearl River estuary, south China. Sci. Total Environ. , 636, 1009-1019.

708

https://doi.org/10.1016/j.scitotenv.2018.04.358

709

Maluping, R.P.P., Lavilla-Pitogo, C.R.R., DePaola, A., Janda, J.M.M., Krovacek, K., Greko,

710

C., 2005. Antimicrobial susceptibility of Aeromonas spp., Vibrio spp. and Plesiomonas

711

shigelloides isolated in the Philippines and Thailand. Int. J. Antimicrob. Agents., 25,

712

348-349. https://doi.org/10.1016/j.ijantimicag.2005.01.003

713

Matsuyama, R., Kuninaga, N., Morimoto, T., Shibano, T., Sudo, A., Sudo, K., Asano, M.,

714

Suzuki, M., Asai, T., 2015. Isolation and antimicrobial susceptibility of Plesiomonas

715

shigelloides from great cormorants (Phalacrocorax carbo hanedae) in Gifu and Shiga

716

Prefectures, Japan. J. Vet. Med. Sci. 77, 1179–1181. https://doi.org/10.1292/jvms.15-

717

0014

718

McCusker, M.P., Alves Ferreira, D., Cooney, D., Martins Alves, B., Fanning, S., Pagès, J.M.,

719

Martins, M., Davin-Regli, A., 2019. Modulation of antimicrobial resistance in clinical

720

isolates of Enterobacter aerogenes: A strategy combining antibiotics and

721

chemosensitisers. J. Glob. Antimicrob. Resist., 16, 187-198.

722

https://doi.org/10.1016/j.jgar.2018.10.009

723

Medema, G., Schets, C., 1993. Occurrence of Plesiomonas shigelloides in surface water:

724

relationship with faecal pollution and trophic state. Zentralbl Hyg Umweltmed., 194,

725

398-404. PMID:8397688

726

Meletis, G., 2016. Carbapenem resistance: overview of the problem and future perspectives.

727

Ther. Adv. Infect. Dis., 3(1), 15-21. https://doi.org/10.1177/2049936115621709

728

Meng, S., Xu, J., Xiong, Y., Ye, C., 2012. Rapid and Sensitive Detection of Plesiomonas

729

shigelloides by Loop-Mediated Isothermal Amplification of the hugA Gene. PLoS One

730

7, 1–6. https://doi.org/10.1371/journal.pone.0041978

731

Mohanta, T., Goel, S., 2014. Prevalence of antibiotic-resistant bacteria in three different

732

aquatic environments over three seasons. Environ. Monit. Assess. 186, 5089–5100.

733

https://doi.org/10.1007/s10661-014-3762-1

734 735 736

Newsom, R., Gallois, C., 1982. Diarrheal disease caused by Plesiomonas shigelloides. Clin. Microbiol. Newsl. 4, 158–159. https://doi.org/10.1016/S0196-4399(82)80049-4 Nolte, F.S.S., Poole, R.M.M., Murphy, G.W.W., Clark, C., Panner, B.J.J., 1988. Proctitis and

737

fatal septicemia caused by Plesiomonas shigelloides in a bisexual man. J. Clin.

738

Microbiol. 26, 388–391.

739

Novoa-Farías, O., Frati-Munari, A.C., Peredo, M.A., Flores-Juárez, S., Novoa-García, O.,

740

Galicia-Tapia, J., Romero-Carpio, C.E., 2016. Susceptibility to rifaximin and other

741

antimicrobial agents of bacteria isolated from acute gastrointestinal infections in

742

Mexico. Rev. Gastroenterol. México (English Ed. 81, 3–10.

743

https://doi.org/10.1016/j.rgmxen.2015.12.001

744

Nwokocha, A.R.C., and Onyemelukwe, N.F., 2014. Plesiomonas shigelloides diarrhea in

745

Enugu area of south eastern Nigeria : Incidence , clinical and epidemiological features

746

13, 68–73. https://doi.org/10.9790/0853-13426873

747

Obi, C.L., Potgieter, N., Bessong, P.O., Coker, A.O., Epoke, J. and, Ndip, R.N., 1995.

748

Aeromonas and Plesiomonas species as bacterial agents of diarrhoea in urban and rural

749

areas of Nigeria: Antibiogram of isolates. Cent. Afr. J. Med., 41, 397-403.

750

PMID:8907605

751

Oxley, A.P.A., Shipton, W., Owens, L., McKay, D., 2002. Bacterial flora from the gut of the

752

wild and cultured banana prawn, Penaeus merguiensis. J. Appl. Microbiol., 93, 214-223.

753

https://doi.org/10.1046/j.1365-2672.2002.01673.x

754

Ozdemir, O., Sari, S., Terzioglu, S., Zenciroglu, A., 2010. Plesiomonas shigelloides Sepsis

755

and Meningoencephalitis in a Surviving Neonate. J. Microbiol. Immunol. Infect. 43,

756

344–346. https://doi.org/10.1016/S1684-1182(10)60053-9

757

Pan, L., Liu, S., Cheng, X., Tao, Y., Yang, T., Li, P., Wang, Z., Shao, D., Zhang, D., 2017.

758

Isolation, identification and genomic analysis of Plesiomonas shigelloides Isolated from

759

diseased Percocypris pingi (Tchang, 1930). Am. J. Biochem. Biotechnol. 13, 226–232.

760

https://doi.org/10.3844/ajbbsp.2017.226.232

761

Papp-Wallace, K.M., Endimiani, A., Taracila, M.A., Bonomo, R.A., 2011. Carbapenems:

762

Past, present, and future. Antimicrob. Agents Chemother. 55, 4943–4960.

763

https://doi.org/10.1128/AAC.00296-11

764

Patel, S., Gandhi, D., Mehta, V., Bhatia, K., Epelbaum, O., 2016. Plesiomonas shigelloides :

765

an extremely rare cause of spontaneous bacterial peritonitis. Acta Gastroenterol. Belg. ,

766

79, 52-53. PMID:26852764

767

Pathak, A., Custer, J.R., Levy, J., 1983. Neonatal septicemia and meningitis due to

768

Plesiomonas shigelloides. Pediatrics 71, 389–391.

769

https://pediatrics.aappublications.org/content/71/3/389.abstract

770

Paul, R., Siitonen, A., Karkkainen, P., 1990. Plesiomonas shigelloides bacteremia in a

771

healthy girl with mild gastroenteritis. J. Clin. Microbiol. 28, 1445-1446.

772

https://jcm.asm.org/content/jcm/28/6/1445.full.pdf

773

Pence, M.A., 2016. Wound Infection with Plesiomonas shigelloides following a freshwater

774

injury. J. Clin. Microbiol. 54, 1180–1182. https://doi.org/10.1128/JCM.02652-15

775

Petrušić, M., Vidaković, D.O., Lazić, S., Radnović, D., Knežević, P., 2018. Prevalence and

776

genetic variability of Plesiomonas shigelloides in temperate climate surface waters of

777

the Pannonian Plain. Arch. Biol. Sci. 70, 99–108.

778

https://doi.org/10.2298/ABS170530029P

779

Pfeiffer, M.L., DuPont, H.L., Ochoa, T.J., 2012. The patient presenting with acute dysentery -

780

A systematic review. J. Infect. 64, 374–386. https://doi.org/10.1016/j.jinf.2012.01.006

781

Proia, L., Anzil, A., Borrego, C., Farrè, M., Llorca, M., Sanchis, J., Bogaerts, P., Balcázar,

782

J.L., Servais, P., 2018a. Occurrence and persistence of carbapenemases genes in hospital

783

and wastewater treatment plants and propagation in the receiving river. J. Hazard. Mater.

784

, 358, 33-43. https://doi.org/https://doi.org/10.1016/j.jhazmat.2018.06.058

785

Proia, L., Anzil, A., Subirats, J., Borrego, C., Farrè, M., Llorca, M., Balcázar, J.L., Servais,

786

P., 2018b. Antibiotic resistance along an urban river impacted by treated wastewaters.

787

Sci. Total Environ. 628–629, 453–466. https://doi.org/10.1016/j.scitotenv.2018.02.083

788

Rautelin, H., Sivonen, A., Kuikka, A., Renkonen, O.V., Valtonen, V., Kosunen, T.U., 1995.

789

Enteric Plesiomonas shigelloides infections in finnish patients. Scand. J. Infect. Dis. 27,

790

495–498. https://doi.org/10.3109/00365549509047052

791 792 793

Riley, P.A., Parasakthi, N., Abdullah, W.A., 1996. Plesiomonas shigelloides bacteremia in a child with leukemia. Clin. Infect. Dis. 23, 206–207. Rizzo, L., Della Sala, A., Fiorentino, A., Li Puma, G., 2014. Disinfection of urban

794

wastewater by solar driven and UV lamp - TiO2 photocatalysis: Effect on a multi drug

795

resistant Escherichia coli strain. Water Res., 53, 145-152.

796

https://doi.org/10.1016/j.watres.2014.01.020

797

Rolston, K.V.I., Hopfer, R.L.O.Y.L., Rolstoni, K.V.I., Hopfer, R.L.O.Y.L., 1984. Diarrhea

798

Due to Plesiomonas shigelloides in Cancer Patients. J. Clin. Microbiol. 20, 597–598.

799

https://jcm.asm.org/content/jcm/20/3/597.full.pdf

800

Roth, T., Hentsch, C., Erard, P., Tschantz, P., 2002. Pyosalpinx: Not always a sexual

801

transmitted disease? Pyosalpinx cuased by Plesiomonas shigelloides in an

802

immunocompetent host. Clin. Microbiol. Infect. 8, 803–805.

803

https://doi.org/10.1046/j.1469-0691.2002.00443.x

804

Rutala, W.A., Sarubbi, F.A., Finch, C.S., Maccormack, J.N., Steinkraus, G.E., 1982. Oyster-

805

associated outbreak of diarrhoeal disease possibly caused by Plesiomonas shigelloides.

806

Lancet. 1, 739 https://doi.org/10.1016/S0140-6736(82)92647-2

807

Sabri, N.A., Schmitt, H., Van der Zaan, B., Gerritsen, H.W., Zuidema, T., Rijnaarts, H.H.M.,

808

Langenhoff, A.A.M., 2018. Prevalence of antibiotics and antibiotic resistance genes in a

809

wastewater effluent-receiving river in the Netherlands. J. Environ. Chem. Eng.( In Press,

810

Corrected Proof). https://doi.org/10.1016/j.jece.2018.03.004

811

Salerno, a., Čižnár, I., Krovacek, K., Conte, M., Dumontet, S., González-Rey, C., Pasquale,

812

V., Ciznar, I., Krovacek, K., Conte, M., Dumontet, S., Gonzalez-Rey, C., Pasquale, V.,

813

2010. Phenotypic characterization and putative virulence factors of human, animal and

814

environmental isolates of Plesiomonas shigelloides. Folia Microbiol. (Praha). 55, 641–

815

647. https://doi.org/10.1007/s12223-010-0104-8

816

Salerno, A., Delétoile, A., Lefevre, M., Ciznar, I., Krovacek, K., Grimont, P., Brisse, S.,

817

Deletolle, A., Lefevre, M., Ciznar, I., Krovacek, K., Grimont, P., Brisse, S., Del??toile,

818

A., Lefevre, M., Ciznar, I., Krovacek, K., Grimont, P., Brisse, S., Delétoile, A., Lefevre,

819

M., Ciznar, I., Krovacek, K., Grimont, P., Brisse, S., Del??toile, A., Lefevre, M., Ciznar,

820

I., Krovacek, K., Grimont, P., Brisse, S., Delétoile, A., Lefevre, M., Ciznar, I.,

821

Krovacek, K., Grimont, P., Brisse, S., Deletolle, A., Lefevre, M., Ciznar, I., Krovacek,

822

K., Grimont, P., Brisse, S., 2007. Recombining population structure of Plesiomonas

823

shigelloides (Enterobacteriaceae) revealed by multilocus sequence typing. J. Bacteriol.

824

189, 7808–7818. https://doi.org/10.1128/JB.00796-07

825 826

Santos, J.A., Rodríguez-Calleja, J.-M., Otero, A., García-López, M.-L., 2015. Chapter 62 – Plesiomonas, in: Molecular Medical Microbiology. pp. 1111–1123.

827 828

https://doi.org/10.1016/B978-0-12-397169-2.00062-7 Schneider, F., Lang, N., Reibke, R., Michaely, H.J.J., Hiddemann, W., Ostermann, H., 2009.

829

Plesiomonas shigelloides pneumonia. Médecine Mal. Infect. 39, 397–400.

830

https://doi.org/10.1016/j.medmal.2008.11.010

831

Shao, S., Hu, Y., Cheng, J., Chen, Y., 2018. Research progress on distribution, migration,

832

transformation of antibiotics and antibiotic resistance genes (ARGs) in aquatic

833

environment. Crit. Rev. Biotechnol. 38, 1–14.

834

https://doi.org/10.1080/07388551.2018.1471038

835

Shigematsu, M., Kaufmann, M.E., Charlett, A., Niho, Y., Pitt, T.L., 2000. An

836

epidemiological study of Plesiomonas shigelloides diarrhoea among Japanese travellers.

837

Epidemiol. Infect. 125, 523–530. https://doi.org/10.1017/S0950268800004817

838

Terpeluk, C., Goldmann, A., Bartmann, P., Pohlandt, F., 1992. Plesiomonas shigelloides

839

sepsis and meningoencephalitis in a neonate. Eur. J. Pediatr. 151, 499–501.

840

https://doi.org/10.1007/BF01957753

841

Tseng, H.-K., Liu, C.-P., Li, W.-C., Su, S.-C., Lee, C.-M., 2002. Characteristics of

842

Plesiomonas shigelloides infection in Taiwan. J. Microbiol. Immunol. Infect., 35, 47-52.

843

PMID:11950120

844

Tsukamoto, T., Kinoshita, Y., Shimada, T., Sakazaki, R., 1978. Two epidemics of diarrhoeal

845

disease possibly caused by Plesiomonas shigelloides. Epidemiology & Infection, 80,

846

275-280. https://doi.org/10.1017/S0022172400053638

847

Tzanetea, R., Konstantopoulos, K., Xanthaki, A., Kalotychou, V., Spiliopoulou, C.,

848

Michalopoulos, A., Rombos, Y., 2002. Plesiomonas shigelloides sepsis in a thalassemia

849

intermedia patient. Scand. J. Infect. Dis., 34, 687-689.

850

https://doi.org/10.1080/00365540210147877

851

Van Damme, L.R.R., Vandepitte, J., 1980. Frequent isolation of Edwardsiella tarda and

852

Plesiomonas shigelloides from healthy Zairese freshwater fish: a possible source of

853

sporadic diarrhea in the tropics. Appl. Environ. Microbiol. 39, 475–479.

854

https://aem.asm.org/content/aem/39/3/475.full.pdf

855

Van Houten, R., Farberman, D., Norton, J., Ellison, J., Kiehlbauch, J., Morris, T., Smith, P.,

856

Houten, R., Farberman, D., Norton, J., Ellison, J., Kiehlbauch, J., Morris, T., Smith, P.,

857

1998. Plesiomonas shigelloides and Salmonella serotype Hartford infections associated

858

with a contaminated water supply - Livingston County, New York, 1996 (Reprinted

859

from MMWR, vol 47, pg 394-396, 1998). Infect. Med. 15, 495–497.

860

https://www.cdc.gov/mmwr/preview/mmwrhtml/00052844.htm

861

Van Loon, F.P.L., Rahim, Z., Chowdhury, K.A., Kay, B.A., Rahman, S.A., 1989. Case report

862

of Plesiomonas shigelloides-associated persistent dysentery and pseudomembranous

863

colitis. J. Clin. Microbiol., 27, 1913-1915.

864

https://jcm.asm.org/content/jcm/27/8/1913.full.pdf

865

Visitsunthorn, N., Komolpis, P., 1995. Antimicrobial therapy in Plesiomonas shigelloides-

866

associated diarrhea in Thai children. Southeast Asian J. Trop. Med. Public Health, 26,

867

86-86. https://www.tm.mahidol.ac.th/seameo/1995-26-1/1995-26-1-86.pdf

868

Wadström, T., Ljungh, Å., 1991. Aeromonas and Plesiomonas as food- and waterborne

869

pathogens. Int. J. Food Microbiol., 12, 303-311. https://doi.org/10.1016/0168-

870

1605(91)90144-E

871

Waecker, N.J., Davis, C.E., Bernstein, G., Spector, S.A., 1988. Plesiomonas shigelloides

872

septicemia and meningitis in a newborn. Pediatr. Infect. Dis. J. 7, 877–879.

873

https://doi.org/10.1097/00006454-198812000-00009

874

Wan, M.T., Chou, C.C., 2015. Class 1 integrons and the antiseptic resistance gene (qacE∆1)

875

in municipal and swine slaughterhouse wastewater treatment plants and wastewater-

876

associated methicillin-resistant staphylococcus aureus. Int. J. Environ. Res. Public

877

Health 12, 6249–6260. https://doi.org/10.3390/ijerph120606249

878

Wong, T.Y., Tsui, H.Y., So, M.K., Lai, J.Y., Lai, S.T., Tse, C.W., Ng, T.K., 2000.

879

Plesiomonas shigelloides infection in Hong Kong: retrospective study of 167 laboratory-

880

confirmed cases. Hong Kong Med. J. 6, 375–380.

881

https://pdfs.semanticscholar.org/5a37/d04903e4627ac1d4ed1846c4a079deabf157.pdf

882

Wouafo, M., Pouillot, R., Kwetche, P.F., Tejiokem, M.-C., Kamgno, J., Fonkoua, M.-C.,

883

2006. An acute foodborne outbreak due to Plesiomonas shigelloides in Yaounde,

884

Cameroon. Foodborne Pathog. Dis., 3, 209-211. https://doi.org/10.1089/fpd.2006.3.209

885

Xia, F.-Q.Q., Liu, P.-N.N., Zhou, Y.-H.H., 2015. Meningoencephalitis caused by

886

Plesiomonas shigelloides in a Chinese neonate: case report and literature review. Ital. J.

887

Pediatr. 41, 3. https://doi.org/10.1186/s13052-014-0107-1

888

Xu, J., Xu, Y., Wang, H., Guo, C., Qiu, H., He, Y., Zhang, Y., Li, X., Meng, W., 2015.

889

Occurrence of antibiotics and antibiotic resistance genes in a sewage treatment plant and

890

its effluent-receiving river. Chemosphere 119, 1379–1385.

891

https://doi.org/10.1016/j.chemosphere.2014.02.040

892

Zhang, Y., Gu, A.Z., Cen, T., Li, X., He, M., Li, D., Chen, J., 2018. Sub-inhibitory

893

concentrations of heavy metals facilitate the horizontal transfer of plasmid-mediated

894

antibiotic resistance genes in water environment. Environ. Pollut. 237, 74–82.

895

https://doi.org/10.1016/j.envpol.2018.01.032

896 897

Zhu, Y.G., Zhao, Y., Li, B., Huang, C.L., Zhang, S.Y., Yu, S., Chen, Y.S., Zhang, T., Gillings, M.R., Su, J.Q., 2017. Continental-scale pollution of estuaries with antibiotic

898

resistance genes. Nat. Microbiol., 2, 16270. https://doi.org/10.1038/nmicrobiol.2016.270

899

Table 1: Descriptive presentation of antibiotic susceptibility profile of freshwater P. shigelloides isolates (N = 182). Recommended antibiotics

Non-recommended antibiotics

Antibiotics

No. R (%)

No. I (%)

No. S (%)

Antibiotics

No. R (%)

No. I (%)

No. S (%)

CRO30C

54 (29.67)

7 (3.85)

122 (67.03)

CZ30C

89 (48.90)

52 (28.57)

42 (23.08)

CAZ30C

62 (34.07)

9 (4.95)

112 (61.54)

ATM30C

66 (36.26)

11 (6.04)

105 (57.69)

TS25C

19 (10.44)

3 (1.65)

161 (88.46)

T30C

31 (17.03)

5 (2.75)

147 (80.77)

LEV5C

5 (2.75)

14 (7.69)

164 (90.11)

GM10C

6 (3.30)

43 (23.64)

133 (73.08)

CIP5C

33 (18.13)

30 (16.48)

120 (65.93)

*PB300C

41 (22.53)

0 (0)

141 (77.47)

PTZ110C

16 (8.79)

9 (4.95)

158 (86.81)

VA30C

163 (89.56)

0 (0)

20 (10.99)

CPM30C

58 (31.87)

4 (2.20)

121 (66.48)

E15C

163 (89.56)

3 (1.65)

17 (9.34)

MEM10C

2 (1.10)

17 (9.34)

164 (90.11)

AK30C

6 (3.30)

53 (29.12)

123 (67.58)

CXM5C

64 (35.17)

42 (23.08)

77 (42.31)

AP10C

103 (56.59)

0 (0)

79 (43.41)

FOX30C

69 (37.91)

11 (6.04)

103 (56.59)

SMX25C

172 (94.51)

5 (2.75)

6 (3.30)

NOR10C

8 (4.40)

8(4.40)

167 (91.76)

C10C

61 (33.52)

0 (0)

121 (66.48)

ETP10C

9 (4.95)

5 (2.75)

169 (92.86)

IMI10C

1 (0.55)

9 (4.95)

173 (95.06)

900

Footnote: *disc diffusion screening was not recommended, for that reason it was grouped among nonRAs; zone interpretation was done using E.

901

coli ATCC®b 25922 (CLSI, 2018) quality control interpretative zone as surrogate.

902 903

904

Table 2: Resistance patterns of freshwater Plesiomonas isolates from the Tyhume, Kubusie and Kat rivers. TY Resistance patterns

TY1

No resistance

2

2

KB TY3

TY4

TY5/6

1

5

9

7

1

KB KB2

3

KB

KT

KT

KB4

5

1

2

KT3

KT 4

KT5

su m

9

4

3

7

4

2

10

63

3

1

0.55

% 34.4

CPM30C

1

CRO30C

1

0.55

ETP10C

1 1

1

0.55

MEM10C

1

1

0.55

NOR10C

1

1

0.55

1

0.55

2

1.09

3

1.64

4

2.19

PTZ110C

1

TS25C

1

CXM5C

1

CAZ30C

1

1 1

1

1

1

CIP5C

1

FOX30C

4

1

2

1

1

1

CAZ30C/CIP5C CIP5C/FOX30C

1

CIP5C/FOX30C CPM30C/NOR10C

CIP5C/CPM30C/CXM5C

2.19 9.29

1

1

0.55

1

1

0.55

1

1

0.55

1

0.55

1

0.55

1

0.55

1

1

0.55

1

0.55

1

3

1.64

3

10

5.47

1

0.55

1

0.55

1

0.55

1 1

CXM5C/FOX30C

CAZ30C/LEV5C/CIP5C

4 17

1

TS25C/CIP5C

CAZ30C/CPM30C/CXM5C

1 5

1

CXM5C/ETP10C

CAZ30C/CPM30C

2 2

1

CRO30C/CAZ30C

PTZ110C/FOX30C

1

1 2

1

4

1 1 1

CIP5C/CXM5C/FOX30C CIP5C/PTZ110C/MEM10C

1 1

CPM30C/CXM5C/FOX30C

1

CRO30C/CXM5C/ETP10C

1

CRO30C/CXM5C/FOX30C

1

TS25C/CIP5C/NOR10C CRO30C/CAZ30C/CPM30C

1

CAZ30C/CPM30C/CXM5C/FOX30C CAZ30C/CXM5C/NOR10C/ETP10C

0.55

0.55

1

2

1.09

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

4

2.19

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

1

0.55

3

1.64

8

4.37

1

0.55

1 1

CRO30C/CPM30C/CXM5C/FOX30C

1 1

CRO30C/LEV5C/CIP5C/FOX30C

1

CRO30C/CAZ30C/CPM30C/CXM5C

1

1

1

1

1

CAZ30C/TS25C/CPM30C/CXM5C/FOX30C

1

CRO30C/CAZ30C/CIP5C/CPM30C/FOX30C

1

0.55

CRO30C/CAZ30C/LEV5C/CPM30C

CAZ30C/TS25C/CIP5C/CPM30C/FOX30C

0.55

1

1

CRO30C/CXM5C/NOR10C/ETP10C

1

1

1

CRO30C/CAZ30C/PTZ110C/CPM30C

0.55 0.55

1

1

CRO30C/CAZ30C/CPM30C/ETP10C

1 1

1

CRO30C/CAZ30C/LEV5C/CPM30C/CXM5C/FOX30C/NOR10C/ETP1 0C

1

CRO30C/CAZ30C/TS25C/CPM30C/CXM5C

1

CRO30C/CAZ30C/TS25C/LEV5C/CIP5C CRO30C/CAZ30C/CIP5C/CPM30C/CXM5C

1 1

1

CRO30C/CAZ30C/CPM30C/CXM5C/FOX30C CRO30C/CAZ30C/CIP5C/CPM30C/NOR10C/ETP10C

1

1

1 1

2

1

CRO30C/CAZ30C/CIP5C/PTZ110C/CXM5C/FOX30C

1

CRO30C/CAZ30C/TS25C/CIP5C/CPM30C/CXM5C CRO30C/CAZ30C/PTZ110C/CPM30C/CXM5C/FOX30C CRO30C/CAZ30C/TS25C/CPM30C/CXM5C/FOX30C

2

1 1

1

1

1

0.55

1

1

0.55

1

2

1.09

2

1.09

CRO30C/CAZ30C/CIP5C/CPM30C/CXM5C/FOX30C

2

CRO30C/CAZ30C/CIP5C/CPM30C/CXM5C/FOX30C/NOR10C

1

1

1

5

2.73

1

0.55

CRO30C/CAZ30C/TS25C/CIP5C/CPM30C/CXM5C/FOX30C

1

1

0.55

CRO30C/CAZ30C/CIP5C/PTZ110C/CPM30C/CXM5C/FOX30C

2

2

1.09

1

5

2.73

CRO30C/CAZ30C/TS25C/CIP5C/PTZ110C/CPM30C/CXM5C/FOX30C

CRO30C/CAZ30C/TS25C/PTZ110C/CPM30C/CXM5C/FOX30C

1

1

0.55

CRO30C/CAZ30C/TS25C/PTZ110C/CPM30C/CXM5C/FOX30C/ETP10C

1

1

0.55

Number of resistance patterns

1

1

8

4

4

5

1

10

5

6

1

4

6

3

8

12

13

7

17

905

i Antibiotics: ceftriaxone (CRO30C), ceftazidime (CAZ30C), trimethoprim-sulfamethoxazole (TS25C), levofloxacin (LEV5C), ciprofloxacin

906

(CIP5C), piperacillin/tazobactam (PTZ110C), cefepime (CPM30C), meropenem (MEM10C), cefuroxime (CXM5C, 5 µg), cefoxitin (FOX30C),

907

norfloxacin

(NOR10C),

ertapenem

(ETP10C),

and

imipenem

(IMI10C).

908

909 910 911 912 913 914 915

Figure1.

916 917 918 919

Figure 2.

920

921

922 923

Figure 3.

924 925 926 927 928

Figure 4.

929

Figure captions

930

Figure 1. Map of the Amathole District Municipality showing the sampling points.

931

Tyhume river points = T1: TH1; T2: TH2; T3: TH3; T4: TH4; T5: TH5; T3a: TH3 water

932

abstraction point; T3d: TH3 domestic sewer inflow; T5m: TH5 manhole; Kat river points =

933

K1: KT1; K2: KT2; K3: KT3; K4: KT4; K5: KT5; K5a: KT5 dumpsite; K4f: KT4 farms;

934

K4f1: KT4 farms1; K4f2: KT4farms2; K4f3: KT4 farms3; K1w: KT1 wastewater treatment

935

plant effluent inflow point; K1e: KT1 wastewater treatment plant; Kubusie river points =

936

B1: KB1; B2: KB2; B3: KB3; B4: KB4; B5: KB5; B5f1: KB5farm1; B5f2: KB5 farm2; B5p:

937

KB5 poultary; B4s: KB4 settlement area; B2f: KB2 farms; B1f1: KB1 farm; B1f2: KB1

938

farm1; and B1f3: KB1 farm2.

939 940

Figure 2. A representative gel for molecular confirmation of P. shigelloides

941

isolates. Line 1= 1kb ladder; line 2 = negative control; 3 to 12 isolates; line 13 = positive

942

control.

943

Figure 3. Heatmap cluster analysis of Plesiomonas isolates’ antibiogram profiles. (A):

944

Plesiomonas isolates from Tyhume River; (B): Plesiomonas isolates from Kubusie River, and

945

(C): Plesiomonas isolates from Kat River. Colour interpretation, Blue (1): resistant, White

946

(2): intermediate and red (3): susceptible. The sampling site is denoted by the first 3 digits of

947

the strains’ names (e.g. KT4). Column and row clusters grouped antibiotics and isolates based

948

on

949

(CAZ30C), trimethoprim-sulfamethoxazole (TS25C), levofloxacin (LEV5C), ciprofloxacin

950

(CIP5C), piperacillin/tazobactam (PTZ110C), cefepime (CPM30C), meropenem (MEM10C),

951

cefuroxime (CXM5C), cefoxitin (FOX30C), norfloxacin (NOR10C), ertapenem (ETP10C),

952

and imipenem (IMI10C).

activity/response

respectively.

Antibiotics:

ceftriaxone

(CRO30C),

ceftazidime

953 954 955 956

Figure 4. Comparison of antibiotic resistance index (ARI) across sampling sites according

957

to recommended (RAs) and non-recommended antibiotics (nonRAs).

(A)

(B)

(C)

Highlights • Freshwater P. shigelloides isolates were tested against 24 antibiotics. • P. shigelloides had co-resistance against cephalosporin, quinolones & fluoroquinolones. • About 95.6% isolates had multiresistance index >0.2 to non-recommended antibiotics. • Empirical treatment of infections model linked 34.07% fatal case risk to choice As. • Environmental model identified pollution risk in 8 out of 15 sites.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: