Accepted Manuscript

Title The identification typing and antimicrobialsusceptibility of Pseudomonas aeruginosa isolated from minkwith hemorrhagic pneumonia

Author Jing Qi Lulu Li Yijun Du Shourong Wang JinwenWang Yanbo Luo Jie Che Jinxing Lu Hui Liu Guangchun HuJixia Li Yanwen Gong Guisheng Wang Ming Hu ShiganYanYuqing Liu Deli Shi

PII S0378-1135(14)00115-1DOI httpdxdoiorgdoi101016jvetmic201402025Reference VETMIC 6527

To appear in VETMIC

Received date 26-11-2013Revised date 10-2-2014Accepted date 13-2-2014

Please cite this article as Qi J Li L Du Y Wang S Wang J Luo Y CheJ Lu J Liu H Hu G Li J Gong Y Wang G Hu M Liu Y Shi DTheidentification typing and antimicrobial susceptibility of Pseudomonas aeruginosaltigtisolated from mink with hemorrhagic pneumonia ltigtVeterinary Microbiologyltigt(2014) httpdxdoiorg101016jvetmic201402025

This is a PDF file of an unedited manuscript that has been accepted for publicationAs a service to our customers we are providing this early version of the manuscriptThe manuscript will undergo copyediting typesetting and review of the resulting proofbefore it is published in its final form Please note that during the production processerrors may be discovered which could affect the content and all legal disclaimers thatapply to the journal pertain

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The identification typing and antimicrobial susceptibility of Pseudomonas aeruginosa 1

isolated from mink with hemorrhagic pneumonia 2

Jing Qiab

Lulu Lia Yijun Du

ab Shourong Wang

c Jinwen Wang

ab Yanbo Luo

a Jie Che

d 3

Jinxing Lud Hui Liu

e Guangchun Hu

e Jixia Li

f Yanwen Gong

f Guisheng Wang

aj Ming Hu

b 4

ShiganYanb Yuqing Liu

ab Deli Shi

a 5

aCollege of Life Sciences Shandong University Jinan 250100

6

bShandong Key Laboratory of Animal Disease Control and Breeding Institute of Animal

7

Science and Veterinary Medicine Shandong Academy of Agricultural Sciences Jinan 250100 8

cCollege of Agricultural Sciences Liaocheng University Liaocheng 252059

9

dNational institute for communicable disease control and prevention Chinese center for

10

disease control and prevention Beijing 102206 11

eJinan municipal center for disease control amp prevention Jinan 250021

12

fJinan military general hospital Jinan 250031

13

jShandong provincial center for animal disease control and prevention Jinan 250022

14

15

Corresponding author 16

Yuqing Liu Mailing address Shandong Key Laboratory of Animal Disease Control and 17

Breeding Institute of Animal Science and Veterinary Medicine Shandong Academy of 18

Agricultural Sciences 8 Sangyuan Road Jinan 250100 China Tel 86-531-88962860 Fax 19

86-531-88967665 E-mail address 13853183199163com 20

Deli Shi Mailing address College of Life Sciences Shandong University 27 Shandanan 21

Road Jinan 250100 China Tel 86-531-88364935 Fax 86-531-88967665 E-mail address 22

dshisdueducn 23

24

25

Revised manuscript without changes marked (clean version)

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Abstract 26

The biological characteristics and molecular epidemiology of Pseudomonas aeruginosa 27

associated with mink hemorrhagic pneumonia from Shandong province of eastern China were 28

determined in this study From 2010 to 2011 30 mink P aeruginosa isolates were identified 29

from lung fecal and feed samples of clinical cases and subjected to serotyping antimicrobial 30

susceptibility testing and pulsed-field gel electrophoresis (PFGE) using SpeI The P 31

aeruginosa isolates belonged to four serotypesmdash21 of type G four of type I three of type M 32

one of type B and one non-typable strain The strains were divided into four large groups as 33

determined by PFGE Isolates from the group 2 were highly homologous and were obtained 34

from the same region as an epidemic All of the isolates were sensitive to piperacillin 35

piperacillintazobactam ceftazidime cefepime imipenem amikacin gentamicin and 36

tobramycin and resistant to ampicillin cefuroxime and cefuroxime axetil A high frequency of 37

resistance was found to ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin 38

and trimethoprimsulfamethoxazole (967) Resistance to ticarcillinclavulanic acid 39

ciprofloxacin and levofloxacin was less common (133) There was no relationship between 40

antibiotic resistance and serotype distribution of the isolates The epidemic serotype of P 41

aeruginosa from the mink hemorrhagic pneumonia in Shandong province was type G which 42

was a clone of commonly found in this province These findings reveal the genetic similarities 43

and antimicrobial susceptibility profiles of P aeruginosa from clinical cases of mink 44

hemorrhagic pneumonia and will facilitate the prevention and control of the disease in 45

Shandong province of China 46

Keywords Mink Pseudomonas aeruginosa Identification Serotyping Antimicrobial 47

susceptibility Pulsed-field gel electrophoresis 48

49

50

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1 Introduction 51

Pseudomonas aeruginosa (P aeruginosa) is a Gram-negative opportunistic pathogen that 52

commonly infects patients with impaired immune responses as hospital-acquired pneumonia 53

(Salomonsen et al 2013) In addition to being a human pathogen P aeruginosa also causes 54

serious harms to dogs cats and fur-bearing animals such as mink fox and raccoons (Lin et al 55

2012 Hariharan et al 2006 Shimizu et al 1974) P aeruginosa has been a major cause of 56

hemorrhagic pneumonia in mink for the last 50 years and the associated mortality is 1 to 57

50 (Knox 1953 Honda et al 1997) The disease is almost exclusively seasonal (from 58

September to early December) and is characterized by sudden deaths Moreover the dead 59

minks are typically found with blood around the nostrils and mouth and severe pathological 60

changes in the lungs (Knox 1953) P aeruginosa is widespread in mink and fox farm 61

surroundings contaminating their water cages water cups and feed troughs (Gierloslashff 1980) 62

The reason why mink are susceptible to this organism remains unclear 63

Three antibioticsmdashβ-lactams aminoglycosides and fluoroquinolonesmdashare commonly used 64

in the treatment of P aeruginosa infections in humans However P aeruginosa resistance to 65

these antibiotics is increasingly reported (Tam et al 2010) Alarmingly the isolates of the P 66

aeruginosa carrying blaNDM-1 gene was also recently identified (Khajuria et al 2013) 67

However there are few studies on antimicrobial resistance in P aeruginosa isolated from 68

animals We analyzed the susceptibility of mink isolates to fluoroquinolones antibiotics and 69

the associated resistance mechanisms (Gao et al 2011) It was found that 564 of the mink 70

P aeruginosa strains from Rongcheng city in Shandong province were resistant to multiple 71

antibiotics with very high levels of resistance to tetracyclines fluoroquinolones and 72

cephalosporins These isolates had limited resistance to spectinomycin Additionally these P 73

aeruginosa isolates were generally sensitive to aminoglycosides such as gentamicin and 74

amikacin and were completely sensitive to imipenem which has not been used in the 75

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1 Introduction 51

Pseudomonas aeruginosa (P aeruginosa) is a Gram-negative opportunistic pathogen that 52

commonly infects patients with impaired immune responses as hospital-acquired pneumonia 53

(Salomonsen et al 2013) In addition to being a human pathogen P aeruginosa also causes 54

serious harms to dogs cats and fur-bearing animals such as mink fox and raccoons (Lin et al 55

2012 Hariharan et al 2006 Shimizu et al 1974) P aeruginosa has been a major cause of 56

hemorrhagic pneumonia in mink for the last 50 years and the associated mortality is 1 to 57

50 (Knox 1953 Honda et al 1997) The disease is almost exclusively seasonal (from 58

September to early December) and is characterized by sudden deaths Moreover the dead 59

minks are typically found with blood around the nostrils and mouth and severe pathological 60

changes in the lungs (Knox 1953) P aeruginosa is widespread in mink and fox farm 61

surroundings contaminating their water cages water cups and feed troughs (Gierloslashff 1980) 62

The reason why mink are susceptible to this organism remains unclear 63

Three antibioticsmdashβ-lactams aminoglycosides and fluoroquinolonesmdashare commonly used 64

in the treatment of P aeruginosa infections in humans However P aeruginosa resistance to 65

these antibiotics is increasingly reported (Tam et al 2010) Alarmingly the isolates of the P 66

aeruginosa carrying blaNDM-1 gene was also recently identified (Khajuria et al 2013) 67

However there are few studies on antimicrobial resistance in P aeruginosa isolated from 68

animals We analyzed the susceptibility of mink isolates to fluoroquinolones antibiotics and 69

the associated resistance mechanisms (Gao et al 2011) It was found that 564 of the mink 70

P aeruginosa strains from Rongcheng city in Shandong province were resistant to multiple 71

antibiotics with very high levels of resistance to tetracyclines fluoroquinolones and 72

cephalosporins These isolates had limited resistance to spectinomycin Additionally these P 73

aeruginosa isolates were generally sensitive to aminoglycosides such as gentamicin and 74

amikacin and were completely sensitive to imipenem which has not been used in the 75

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veterinary clinics of the study region (personal communication) 76

In order to effectively treat mink hemorrhagic pneumonia it is necessary to understand the 77

antimicrobial susceptibility of related pathogens and epidemiological data In addition to the 78

traditional methods of serotyping pulsed-field gel electrophoresis (PFGE) is considered as the 79

gold standard for P aeruginosa typing (Grundmann et al 1995 Tenover et al 1997 80

Johnson et al 2007) To facilitate clinical treatment we collected lung fecal and feed 81

samples from dead or sick mink with hemorrhagic pneumonia in Shandong province for 82

bacterial isolation identification and antimicrobial susceptibility testing Serotyping and 83

PFGE typing were used to identify epidemic strains and determine the relationship of the 84

isolates obtained from different parts of Shandong province To our best knowledge this is the 85

first report describing mink P aeruginosa in China 86

2 Materials and methods 87

21 Farm selection and sample collection 88

Mink farms in Weihai Linyi Jiaozhou and Jinan Shandong province China were 89

screened for hemorrhagic pneumonia in the autumn of 2010 and 2011 Fecal samples feed 90

samples (frozen chicken intestines) and lung samples from dead or sick mink were collected 91

for bacterial isolation 92

22 Isolation and culture characteristics of P aeruginosa 93

Samples were streaked onto nalidixic acid cetrimide medium (NAC Agar Qingdao Hope 94

Biol-Technology Co Ltd) and blood agar (Qingdao Haibo biotech company) and incubated 95

for 18-20 h at 37degC A single irregular blue-green colony was picked for Gram staining 96

Hemolysis was identified by clearing around the colony on blood agar plates 97

23 Biochemical identification of P aeruginosa 98

Isolates were inoculated onto sterile TSA culture medium and incubated at 37degC for 24 h A 99

bacterial suspension equivalent to 05 McFarland units for each isolate was prepared The 100

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VITEK-2 compact automated microbial identification system (bioMerieux France Bio Co 101

Ltd) was used for physiological and biochemical identification of bacterial isolates 102

24 O-antigen serotyping 103

Serotyping was performed using the slide agglutination method and commercially available 104

polyvalent I II and III group specific antisera against 14 O antigens (Denka Seiken Co Ltd 105

Japan) All isolates were incubated on blood agar plates for 18 h at 37 degC A clean glass slide 106

was prepared with a drop of serum on the sample side and a drop of normal saline on the 107

control side A typical bacterial colony was suspended in normal saline One loop of the dense 108

suspension was placed adjacent to each drop on the slide The slide was tilted back and forth 109

to mix the two drops and observed for signs of agglutination The negative saline control was 110

performed to identify no spontaneous agglutination The positive sample demonstrated 111

agglutination within one minute on the antisera side and no agglutination with the negative 112

saline control (Homma 1982) 113

25 Antimicrobial susceptibility testing 114

Antimicrobial susceptibility testing for all P aeruginosa isolates was performed using the 115

VITEK-2 compact automated microbial identification system and the 116

results were interpreted according to the breakpoints established by the Clinical and 117

Laboratory Standards Institute documents M31-A3 (2008) and M100-S21 (2011) The 118

following antimicrobial agents were tested using the VITEK 2 AST-GN13 cards (bioMerieux 119

France) including β-lactams (ampicillin ampicillinsulbactam piperacillin 120

ticarcillinclavulanic piperacillintazobactam cefazolin cefuroxime cefuroxime axetil 121

cefotetan ceftriaxone ceftazidime cefepime and imipenem) aminoglycosides (amikacin 122

gentamicin and tobramycin) fluoroquinones (ciprofloxacin and levofloxacin) nitrofurantoin 123

and trimethoprimsulfamethoxazole E coli ATCC 25922 and P aeruginosa ATCC 27853 124

were used as quality control strains 125

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26 Pulsed-field gel electrophoresis 126

The PFGE procedure has been described elsewhere (Nauerby et al 2000) and was 127

followed with some modifications for the P aeruginosa isolates Slices (1 mm thick) of DNA 128

embedded in 1 Seakem Gold agarose (SeaKem Gold purchased from the Swiss company 129

Lonza) were digested using SpeI (Takara Biotechnology Co Ltd) for 4 h at 37degC The 130

XbaI-restricted DNA of Salmonella enterica serovar Braenderup H9812 was used as a 131

molecular size marker After restriction digestion fragments were resolved in 1 agarose 132

with 05timesTBE buffer at 6 Vcm at 14degC using a CHEF-DR III pulsed-field electrophoresis 133

system (Bio-Rad Laboratories CA) Pulse times were 5-15 s for 85 h and 15-50 s for an 134

additional 85 h Gels were stained for 30 min with Gelred (Biotium USA) destained in 135

distilled water and photographed in UV-light PFGE patterns were visually assessed and 136

interpreted in accordance with previously published guidelines (Tenover et al 1995) The 137

resulting band profiles were analyzed using Bionumerics software (Applied Maths Incver 138

650) with Dice band based comparison and a position tolerance of 17 (Carrico et al 139

2005) 140

3 Results 141

31 Isolation identification and cultural characteristics of mink P aeruginosa isolates 142

A total of thirty P aeruginosa isolates were obtained from hemorrhagic pneumonia cases in 143

the countryside or the suburbs of different cities in Shandong province The strains included 144

28 lung isolates a feed isolate and a fecal isolate The isolates demonstrated hemolysis 145

around the colonies on blood agar plates (Table 1) Some isolates produced soluble pyocyanin 146

and fluorescein making them green and some strains did not produce pigment 147

32 Biochemical identification of mink P aeruginosa isolates 148

Biochemical evaluation showed that all P aeruginosa isolates were oxidase-positive and 149

hydrolyzed gelatin but not starch They metabolized glucose xylose and fructose but not 150

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sucrose maltose and trehalose The isolates did not produce indole and hydrogen sulfide 151

Methyl red testing was all negative 152

33 Serotype identification of mink P aeruginosa 153

Glass plate agglutination test results are shown in Table 1 P aeruginosa from mink 154

belonged to four serotypes serotype G (2130) I (430) M (330) and B (130) One isolate 155

was non-typable The 21 type G isolates consisted of eight lung isolates and one stool isolate 156

obtained from the Rongcheng region of Weihai city in November 2011 One feed isolate from 157

the same area was type M The remaining four serotypes were obtained from different 158

geographical regions 159

34 Antimicrobial susceptibility of mink P aeruginosa 160

All isolates were sensitive to piperacillin piperacillintazobactam ceftazidime cefepime 161

imipenem amikacin gentamicin and tobramycin and resistant to ampicillin cefuroxime and 162

cefuroxime axetil The number of resistant isolates relative to the total was indicated as A 163

high incidence of resistance was found to ampicillinsulbactam cefazolin cefotetan 164

ceftriaxone nitrofurantoin and trimethoprimsulfamethoxazole (967) A lower percentage 165

of isolates (133) resistant to ticarcillinclavulanic acid ciprofloxacin and levofloxacin were 166

detected 167

35 PFGE typing and cluster analysis 168

Isolates were defined as belonging to the same strain if the isolates had indistinguishable 169

PFGE profiles If the isolates differed by 1 to 5 bands (corresponding to a similarity above 170

85) they were regarded as belonging to a cluster of closely related strains (Tenover et al 171

1997) The PFGE patterns of P aeruginosa we isolated demonstrated a similarity of more 172

than 77 Based on differences found in the dissimilar bands the isolates were divided into 173

four groups (1 2 3 and 4) (Figure 1) The similarity of isolates within group 1 was more than 174

835 The similarity of isolates within group 2 was more than 968 These were lung 175

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isolates with the same G type and isolated from the same geographic location These findings 176

were consistent with spread of a single progenitor clone in this region Group 3 consisted of 177

isolates with more than 774 similarity and several serotypes The similarity of isolates 178

within group 4 was more than 784 with nine type G and two type M isolates There were 179

some isolates with 100 similarity and exactly the identical PFGE band within group 2 3 and 180

4 respectively such as DA~DJ C3F9 and PA+ 11082623 and 111204 11201 and 111202 181

11092617 and 11092618 (marked in the rectangle) 182

4 Discussion 183

Serotype G was the most common serotype among the isolates in accordance with 184

previous observations from the USA and Europe (Habs 1957 Nordstoga 1968 Karlsson et 185

al 1971 Mejerland 1978 Long and Gorham 1981 Elsheik et al 1988 Hammer et al 186

2003) This suggests that P aeruginosa type G plays an important role in mink hemorrhagic 187

pneumonia followed by serotypes I M and B Serotype I is usually found in caprine P 188

aeruginosa isolates and we also found two goat isolates in Shandong province belonging to 189

serotype I which was consistent with the finding in a previous report (Wang 2004) One 190

isolate could not be typed (JZ01) because no specific agglutination was observed after testing 191

with all known serotypes Thus it might represent a new serotype Generally epidemic 192

isolates are usually serotype G with other serotypes occurring sporadically and infrequently 193

The reason why serotype G is so common in mink P aeruginosa isolates needs to be further 194

explored 195

The PFGE patterns demonstrated the regional characteristics of the isolates The isolates 196

inside each cluster were highly homologous especially in the group 2 which were lung 197

isolates with mink hemorrhagic pneumonia from the Rongcheng region of Weihai city In 198

group 4 two isolates (C3sl and QT1) belonged to serotype M and the rest were type G The 199

isolates marked in the rectangle in Figure 1 showed exactly the identical PFGE profile 200

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together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

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tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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VITEK-2 compact automated microbial identification system (bioMerieux France Bio Co 101

Ltd) was used for physiological and biochemical identification of bacterial isolates 102

24 O-antigen serotyping 103

Serotyping was performed using the slide agglutination method and commercially available 104

polyvalent I II and III group specific antisera against 14 O antigens (Denka Seiken Co Ltd 105

Japan) All isolates were incubated on blood agar plates for 18 h at 37 degC A clean glass slide 106

was prepared with a drop of serum on the sample side and a drop of normal saline on the 107

control side A typical bacterial colony was suspended in normal saline One loop of the dense 108

suspension was placed adjacent to each drop on the slide The slide was tilted back and forth 109

to mix the two drops and observed for signs of agglutination The negative saline control was 110

performed to identify no spontaneous agglutination The positive sample demonstrated 111

agglutination within one minute on the antisera side and no agglutination with the negative 112

saline control (Homma 1982) 113

25 Antimicrobial susceptibility testing 114

Antimicrobial susceptibility testing for all P aeruginosa isolates was performed using the 115

VITEK-2 compact automated microbial identification system and the 116

results were interpreted according to the breakpoints established by the Clinical and 117

Laboratory Standards Institute documents M31-A3 (2008) and M100-S21 (2011) The 118

following antimicrobial agents were tested using the VITEK 2 AST-GN13 cards (bioMerieux 119

France) including β-lactams (ampicillin ampicillinsulbactam piperacillin 120

ticarcillinclavulanic piperacillintazobactam cefazolin cefuroxime cefuroxime axetil 121

cefotetan ceftriaxone ceftazidime cefepime and imipenem) aminoglycosides (amikacin 122

gentamicin and tobramycin) fluoroquinones (ciprofloxacin and levofloxacin) nitrofurantoin 123

and trimethoprimsulfamethoxazole E coli ATCC 25922 and P aeruginosa ATCC 27853 124

were used as quality control strains 125

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26 Pulsed-field gel electrophoresis 126

The PFGE procedure has been described elsewhere (Nauerby et al 2000) and was 127

followed with some modifications for the P aeruginosa isolates Slices (1 mm thick) of DNA 128

embedded in 1 Seakem Gold agarose (SeaKem Gold purchased from the Swiss company 129

Lonza) were digested using SpeI (Takara Biotechnology Co Ltd) for 4 h at 37degC The 130

XbaI-restricted DNA of Salmonella enterica serovar Braenderup H9812 was used as a 131

molecular size marker After restriction digestion fragments were resolved in 1 agarose 132

with 05timesTBE buffer at 6 Vcm at 14degC using a CHEF-DR III pulsed-field electrophoresis 133

system (Bio-Rad Laboratories CA) Pulse times were 5-15 s for 85 h and 15-50 s for an 134

additional 85 h Gels were stained for 30 min with Gelred (Biotium USA) destained in 135

distilled water and photographed in UV-light PFGE patterns were visually assessed and 136

interpreted in accordance with previously published guidelines (Tenover et al 1995) The 137

resulting band profiles were analyzed using Bionumerics software (Applied Maths Incver 138

650) with Dice band based comparison and a position tolerance of 17 (Carrico et al 139

2005) 140

3 Results 141

31 Isolation identification and cultural characteristics of mink P aeruginosa isolates 142

A total of thirty P aeruginosa isolates were obtained from hemorrhagic pneumonia cases in 143

the countryside or the suburbs of different cities in Shandong province The strains included 144

28 lung isolates a feed isolate and a fecal isolate The isolates demonstrated hemolysis 145

around the colonies on blood agar plates (Table 1) Some isolates produced soluble pyocyanin 146

and fluorescein making them green and some strains did not produce pigment 147

32 Biochemical identification of mink P aeruginosa isolates 148

Biochemical evaluation showed that all P aeruginosa isolates were oxidase-positive and 149

hydrolyzed gelatin but not starch They metabolized glucose xylose and fructose but not 150

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sucrose maltose and trehalose The isolates did not produce indole and hydrogen sulfide 151

Methyl red testing was all negative 152

33 Serotype identification of mink P aeruginosa 153

Glass plate agglutination test results are shown in Table 1 P aeruginosa from mink 154

belonged to four serotypes serotype G (2130) I (430) M (330) and B (130) One isolate 155

was non-typable The 21 type G isolates consisted of eight lung isolates and one stool isolate 156

obtained from the Rongcheng region of Weihai city in November 2011 One feed isolate from 157

the same area was type M The remaining four serotypes were obtained from different 158

geographical regions 159

34 Antimicrobial susceptibility of mink P aeruginosa 160

All isolates were sensitive to piperacillin piperacillintazobactam ceftazidime cefepime 161

imipenem amikacin gentamicin and tobramycin and resistant to ampicillin cefuroxime and 162

cefuroxime axetil The number of resistant isolates relative to the total was indicated as A 163

high incidence of resistance was found to ampicillinsulbactam cefazolin cefotetan 164

ceftriaxone nitrofurantoin and trimethoprimsulfamethoxazole (967) A lower percentage 165

of isolates (133) resistant to ticarcillinclavulanic acid ciprofloxacin and levofloxacin were 166

detected 167

35 PFGE typing and cluster analysis 168

Isolates were defined as belonging to the same strain if the isolates had indistinguishable 169

PFGE profiles If the isolates differed by 1 to 5 bands (corresponding to a similarity above 170

85) they were regarded as belonging to a cluster of closely related strains (Tenover et al 171

1997) The PFGE patterns of P aeruginosa we isolated demonstrated a similarity of more 172

than 77 Based on differences found in the dissimilar bands the isolates were divided into 173

four groups (1 2 3 and 4) (Figure 1) The similarity of isolates within group 1 was more than 174

835 The similarity of isolates within group 2 was more than 968 These were lung 175

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isolates with the same G type and isolated from the same geographic location These findings 176

were consistent with spread of a single progenitor clone in this region Group 3 consisted of 177

isolates with more than 774 similarity and several serotypes The similarity of isolates 178

within group 4 was more than 784 with nine type G and two type M isolates There were 179

some isolates with 100 similarity and exactly the identical PFGE band within group 2 3 and 180

4 respectively such as DA~DJ C3F9 and PA+ 11082623 and 111204 11201 and 111202 181

11092617 and 11092618 (marked in the rectangle) 182

4 Discussion 183

Serotype G was the most common serotype among the isolates in accordance with 184

previous observations from the USA and Europe (Habs 1957 Nordstoga 1968 Karlsson et 185

al 1971 Mejerland 1978 Long and Gorham 1981 Elsheik et al 1988 Hammer et al 186

2003) This suggests that P aeruginosa type G plays an important role in mink hemorrhagic 187

pneumonia followed by serotypes I M and B Serotype I is usually found in caprine P 188

aeruginosa isolates and we also found two goat isolates in Shandong province belonging to 189

serotype I which was consistent with the finding in a previous report (Wang 2004) One 190

isolate could not be typed (JZ01) because no specific agglutination was observed after testing 191

with all known serotypes Thus it might represent a new serotype Generally epidemic 192

isolates are usually serotype G with other serotypes occurring sporadically and infrequently 193

The reason why serotype G is so common in mink P aeruginosa isolates needs to be further 194

explored 195

The PFGE patterns demonstrated the regional characteristics of the isolates The isolates 196

inside each cluster were highly homologous especially in the group 2 which were lung 197

isolates with mink hemorrhagic pneumonia from the Rongcheng region of Weihai city In 198

group 4 two isolates (C3sl and QT1) belonged to serotype M and the rest were type G The 199

isolates marked in the rectangle in Figure 1 showed exactly the identical PFGE profile 200

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together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

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tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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26 Pulsed-field gel electrophoresis 126

The PFGE procedure has been described elsewhere (Nauerby et al 2000) and was 127

followed with some modifications for the P aeruginosa isolates Slices (1 mm thick) of DNA 128

embedded in 1 Seakem Gold agarose (SeaKem Gold purchased from the Swiss company 129

Lonza) were digested using SpeI (Takara Biotechnology Co Ltd) for 4 h at 37degC The 130

XbaI-restricted DNA of Salmonella enterica serovar Braenderup H9812 was used as a 131

molecular size marker After restriction digestion fragments were resolved in 1 agarose 132

with 05timesTBE buffer at 6 Vcm at 14degC using a CHEF-DR III pulsed-field electrophoresis 133

system (Bio-Rad Laboratories CA) Pulse times were 5-15 s for 85 h and 15-50 s for an 134

additional 85 h Gels were stained for 30 min with Gelred (Biotium USA) destained in 135

distilled water and photographed in UV-light PFGE patterns were visually assessed and 136

interpreted in accordance with previously published guidelines (Tenover et al 1995) The 137

resulting band profiles were analyzed using Bionumerics software (Applied Maths Incver 138

650) with Dice band based comparison and a position tolerance of 17 (Carrico et al 139

2005) 140

3 Results 141

31 Isolation identification and cultural characteristics of mink P aeruginosa isolates 142

A total of thirty P aeruginosa isolates were obtained from hemorrhagic pneumonia cases in 143

the countryside or the suburbs of different cities in Shandong province The strains included 144

28 lung isolates a feed isolate and a fecal isolate The isolates demonstrated hemolysis 145

around the colonies on blood agar plates (Table 1) Some isolates produced soluble pyocyanin 146

and fluorescein making them green and some strains did not produce pigment 147

32 Biochemical identification of mink P aeruginosa isolates 148

Biochemical evaluation showed that all P aeruginosa isolates were oxidase-positive and 149

hydrolyzed gelatin but not starch They metabolized glucose xylose and fructose but not 150

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sucrose maltose and trehalose The isolates did not produce indole and hydrogen sulfide 151

Methyl red testing was all negative 152

33 Serotype identification of mink P aeruginosa 153

Glass plate agglutination test results are shown in Table 1 P aeruginosa from mink 154

belonged to four serotypes serotype G (2130) I (430) M (330) and B (130) One isolate 155

was non-typable The 21 type G isolates consisted of eight lung isolates and one stool isolate 156

obtained from the Rongcheng region of Weihai city in November 2011 One feed isolate from 157

the same area was type M The remaining four serotypes were obtained from different 158

geographical regions 159

34 Antimicrobial susceptibility of mink P aeruginosa 160

All isolates were sensitive to piperacillin piperacillintazobactam ceftazidime cefepime 161

imipenem amikacin gentamicin and tobramycin and resistant to ampicillin cefuroxime and 162

cefuroxime axetil The number of resistant isolates relative to the total was indicated as A 163

high incidence of resistance was found to ampicillinsulbactam cefazolin cefotetan 164

ceftriaxone nitrofurantoin and trimethoprimsulfamethoxazole (967) A lower percentage 165

of isolates (133) resistant to ticarcillinclavulanic acid ciprofloxacin and levofloxacin were 166

detected 167

35 PFGE typing and cluster analysis 168

Isolates were defined as belonging to the same strain if the isolates had indistinguishable 169

PFGE profiles If the isolates differed by 1 to 5 bands (corresponding to a similarity above 170

85) they were regarded as belonging to a cluster of closely related strains (Tenover et al 171

1997) The PFGE patterns of P aeruginosa we isolated demonstrated a similarity of more 172

than 77 Based on differences found in the dissimilar bands the isolates were divided into 173

four groups (1 2 3 and 4) (Figure 1) The similarity of isolates within group 1 was more than 174

835 The similarity of isolates within group 2 was more than 968 These were lung 175

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isolates with the same G type and isolated from the same geographic location These findings 176

were consistent with spread of a single progenitor clone in this region Group 3 consisted of 177

isolates with more than 774 similarity and several serotypes The similarity of isolates 178

within group 4 was more than 784 with nine type G and two type M isolates There were 179

some isolates with 100 similarity and exactly the identical PFGE band within group 2 3 and 180

4 respectively such as DA~DJ C3F9 and PA+ 11082623 and 111204 11201 and 111202 181

11092617 and 11092618 (marked in the rectangle) 182

4 Discussion 183

Serotype G was the most common serotype among the isolates in accordance with 184

previous observations from the USA and Europe (Habs 1957 Nordstoga 1968 Karlsson et 185

al 1971 Mejerland 1978 Long and Gorham 1981 Elsheik et al 1988 Hammer et al 186

2003) This suggests that P aeruginosa type G plays an important role in mink hemorrhagic 187

pneumonia followed by serotypes I M and B Serotype I is usually found in caprine P 188

aeruginosa isolates and we also found two goat isolates in Shandong province belonging to 189

serotype I which was consistent with the finding in a previous report (Wang 2004) One 190

isolate could not be typed (JZ01) because no specific agglutination was observed after testing 191

with all known serotypes Thus it might represent a new serotype Generally epidemic 192

isolates are usually serotype G with other serotypes occurring sporadically and infrequently 193

The reason why serotype G is so common in mink P aeruginosa isolates needs to be further 194

explored 195

The PFGE patterns demonstrated the regional characteristics of the isolates The isolates 196

inside each cluster were highly homologous especially in the group 2 which were lung 197

isolates with mink hemorrhagic pneumonia from the Rongcheng region of Weihai city In 198

group 4 two isolates (C3sl and QT1) belonged to serotype M and the rest were type G The 199

isolates marked in the rectangle in Figure 1 showed exactly the identical PFGE profile 200

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together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

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tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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sucrose maltose and trehalose The isolates did not produce indole and hydrogen sulfide 151

Methyl red testing was all negative 152

33 Serotype identification of mink P aeruginosa 153

Glass plate agglutination test results are shown in Table 1 P aeruginosa from mink 154

belonged to four serotypes serotype G (2130) I (430) M (330) and B (130) One isolate 155

was non-typable The 21 type G isolates consisted of eight lung isolates and one stool isolate 156

obtained from the Rongcheng region of Weihai city in November 2011 One feed isolate from 157

the same area was type M The remaining four serotypes were obtained from different 158

geographical regions 159

34 Antimicrobial susceptibility of mink P aeruginosa 160

All isolates were sensitive to piperacillin piperacillintazobactam ceftazidime cefepime 161

imipenem amikacin gentamicin and tobramycin and resistant to ampicillin cefuroxime and 162

cefuroxime axetil The number of resistant isolates relative to the total was indicated as A 163

high incidence of resistance was found to ampicillinsulbactam cefazolin cefotetan 164

ceftriaxone nitrofurantoin and trimethoprimsulfamethoxazole (967) A lower percentage 165

of isolates (133) resistant to ticarcillinclavulanic acid ciprofloxacin and levofloxacin were 166

detected 167

35 PFGE typing and cluster analysis 168

Isolates were defined as belonging to the same strain if the isolates had indistinguishable 169

PFGE profiles If the isolates differed by 1 to 5 bands (corresponding to a similarity above 170

85) they were regarded as belonging to a cluster of closely related strains (Tenover et al 171

1997) The PFGE patterns of P aeruginosa we isolated demonstrated a similarity of more 172

than 77 Based on differences found in the dissimilar bands the isolates were divided into 173

four groups (1 2 3 and 4) (Figure 1) The similarity of isolates within group 1 was more than 174

835 The similarity of isolates within group 2 was more than 968 These were lung 175

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isolates with the same G type and isolated from the same geographic location These findings 176

were consistent with spread of a single progenitor clone in this region Group 3 consisted of 177

isolates with more than 774 similarity and several serotypes The similarity of isolates 178

within group 4 was more than 784 with nine type G and two type M isolates There were 179

some isolates with 100 similarity and exactly the identical PFGE band within group 2 3 and 180

4 respectively such as DA~DJ C3F9 and PA+ 11082623 and 111204 11201 and 111202 181

11092617 and 11092618 (marked in the rectangle) 182

4 Discussion 183

Serotype G was the most common serotype among the isolates in accordance with 184

previous observations from the USA and Europe (Habs 1957 Nordstoga 1968 Karlsson et 185

al 1971 Mejerland 1978 Long and Gorham 1981 Elsheik et al 1988 Hammer et al 186

2003) This suggests that P aeruginosa type G plays an important role in mink hemorrhagic 187

pneumonia followed by serotypes I M and B Serotype I is usually found in caprine P 188

aeruginosa isolates and we also found two goat isolates in Shandong province belonging to 189

serotype I which was consistent with the finding in a previous report (Wang 2004) One 190

isolate could not be typed (JZ01) because no specific agglutination was observed after testing 191

with all known serotypes Thus it might represent a new serotype Generally epidemic 192

isolates are usually serotype G with other serotypes occurring sporadically and infrequently 193

The reason why serotype G is so common in mink P aeruginosa isolates needs to be further 194

explored 195

The PFGE patterns demonstrated the regional characteristics of the isolates The isolates 196

inside each cluster were highly homologous especially in the group 2 which were lung 197

isolates with mink hemorrhagic pneumonia from the Rongcheng region of Weihai city In 198

group 4 two isolates (C3sl and QT1) belonged to serotype M and the rest were type G The 199

isolates marked in the rectangle in Figure 1 showed exactly the identical PFGE profile 200

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together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

Page 10 of 15

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10

tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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ted

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isolates with the same G type and isolated from the same geographic location These findings 176

were consistent with spread of a single progenitor clone in this region Group 3 consisted of 177

isolates with more than 774 similarity and several serotypes The similarity of isolates 178

within group 4 was more than 784 with nine type G and two type M isolates There were 179

some isolates with 100 similarity and exactly the identical PFGE band within group 2 3 and 180

4 respectively such as DA~DJ C3F9 and PA+ 11082623 and 111204 11201 and 111202 181

11092617 and 11092618 (marked in the rectangle) 182

4 Discussion 183

Serotype G was the most common serotype among the isolates in accordance with 184

previous observations from the USA and Europe (Habs 1957 Nordstoga 1968 Karlsson et 185

al 1971 Mejerland 1978 Long and Gorham 1981 Elsheik et al 1988 Hammer et al 186

2003) This suggests that P aeruginosa type G plays an important role in mink hemorrhagic 187

pneumonia followed by serotypes I M and B Serotype I is usually found in caprine P 188

aeruginosa isolates and we also found two goat isolates in Shandong province belonging to 189

serotype I which was consistent with the finding in a previous report (Wang 2004) One 190

isolate could not be typed (JZ01) because no specific agglutination was observed after testing 191

with all known serotypes Thus it might represent a new serotype Generally epidemic 192

isolates are usually serotype G with other serotypes occurring sporadically and infrequently 193

The reason why serotype G is so common in mink P aeruginosa isolates needs to be further 194

explored 195

The PFGE patterns demonstrated the regional characteristics of the isolates The isolates 196

inside each cluster were highly homologous especially in the group 2 which were lung 197

isolates with mink hemorrhagic pneumonia from the Rongcheng region of Weihai city In 198

group 4 two isolates (C3sl and QT1) belonged to serotype M and the rest were type G The 199

isolates marked in the rectangle in Figure 1 showed exactly the identical PFGE profile 200

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9

together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

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tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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9

together with the same serotype from the same or the different locations These findings 201

suggest there was spread of the same clone of mink P aeruginosa to different regions of 202

Shandong province Several potential mechanisms for spread exist Mink farms may have a 203

similar food supply generally from frozen fish and fish byproducts as well as other 204

byproducts from slaughtering of poultry swine and cattle mixed with various cereal products 205

Mink farms may also share the same feed kitchens or exchange breeding animals within these 206

cities 207

We isolated one fecal strain C3F9 (group 3) which shared the identical PFGE profile as a 208

type G lung isolate PA+ One P aeruginosa strain C3sl (group 4) from mink feed (frozen 209

chicken meat and animal viscera) had 955 similarity to the lung isolate 02 This suggests 210

that the food chain may play a role in the horizontal transmission of mink P aeruginosa 211

causing hemorrhagic pneumonia It has been reported that feed sinks equipment personnel 212

air and feed production personnel can be sources of outbreaks of hemorrhagic pneumonia in 213

mink (Shimizu et al 1974 Gierloslashff 1980 Hammer et al 2003) 214

Some of the antimicrobial agents tested in our study such as ampicillin 215

ampicillinsulbactam gentamicin ciprofloxacin levofloxacin and 216

trimethoprimsulfamethoxazole are used for treatment in veterinary clinics and hospitals 217

Tobramycin is rarely used in veterinary medicine but rather frequently in human medicine 218

The remaining antimicrobial agents included in the present study are more relevant for use in 219

human medicine Among the 30 isolates of P aeruginosa one was resistant to three 220

antibiotics 25 strains were resistant to nine antibiotics and four strains were resistant to 12 221

antibiotics Multi-drug resistance and cross-resistance were common All strains were 222

resistant to ampicillin cefuroxime and cefuroxime axetil The frequency of resistance to 223

ampicillinsulbactam cefazolin cefotetan ceftriaxone nitrofurantoin and trimethoprim 224

trimethoprimsulfamethoxazole was 967 All strains were sensitive to amikacin gentamicin 225

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10

tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

Page 10 of 15

Accep

ted

Man

uscr

ipt

10

tobramycin and imipenem There are few reports of drug-resistance in bacteria from fur 226

bearing animals A report from Denmark evaluating isolates obtained from 2000 to 2005 227

found 39 mink P aeruginosa isolates were sensitive to gentamicin and polymyxin and 228

resistant to ampicillin ampicillinclavulanic acid cephalothin chloramphenicol lincosamides 229

macrolides and spectinomycin The frequency of resistance to methoxy pyrimidine 230

sulfonamides tetracycline kanamycin and enrofloxacin was 923 897 667 and 51 231

respectively (Pedersen et al 2009) These findings were different from ours likely due to our 232

widespread use of penicillin tetracycline and the 3rd

generation cephalosporin (ie ceftiofur) 233

however relatively less use of aminoglycosides (ie amikacin) and aminocyclitol antibiotics 234

(ie apramycin) used in other animal species but rarely in mink breeding farms in Shandong 235

province (personal communication) Imipenem was prohibited from use in veterinary clinics 236

in China which could explain the total susceptibility of the strains Antibiotics that are known 237

to be effective by antimicrobial susceptibility testing and alternating rotations of medications 238

should be used for the treatment of bacterial infections It is necessary to avoid continuous use 239

of a single drug and to reduce the blind rotation of medications in order to prevent multi-drug 240

resistance and reduce the mortality due to the ineffective antibiotic treatment of P aeruginosa 241

infections While the sample size is small it appears that there was no correlation between 242

antibiotic resistance and serotype However four isolates (11092615 11092616 11092617 243

and 11092618) that exhibited resistance to ticarcillinclavulanic acid ciprofloxacin and 244

levofloxacin were from the same geographic area and shared the same serotype G 245

It is necessary to regularly monitor drug susceptibility and molecular epidemiology of P 246

aeruginosa in mink and explore the potential routes of infection Strains with high virulence 247

and good immunogenicity should be selected to develop vaccines An appropriate antibiotics 248

rotation should be used to provide effective measures for treatment of mink hemorrhagic 249

pneumonia by P aeruginosa 250

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Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

Page 11 of 15

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ted

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uscr

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11

Conflict of interest statement 251

The authors do not have any financial or personal conflicts of interest regarding the work 252

presented in this article 253

Acknowledgements 254

The technical assistance and guidance of Dr Qijing Zhang is grateful acknowledged The 255

project was supported by National Science and Technology major projects sub-topics 256

(2013ZX10004217003) Shandong Provincial Natural Science Foundation (ZR2013CQ037) 257

Research on the key technology of producing superior fur in different ecoregions 258

(200903014-06) Science and Technology Development Program of Shandong Province 259

(2013GNC11032) and Shandong Provincial Agricultural significant application of 260

technological innovation project 261

References 262

Gao C Hu M Qi J Zhu XL Liu CB Shan H Qin XB LiuYQ Gao PJ 2011 263

Resistance and tolerance of Pseudomonas aeruginosa to ciprofloxacin J Shandong 264

University (Health Sciences) 49 38-45 265

Carriccedilo JA Pinto FR Simas C Nunes S Sousa NG Frazatildeo N de Lencastre H 266

Almeida JS 2005 Assessment of band-based similarity coefficients for automatic type 267

and subtype classification of microbial isolates analyzed by pulsed-field gel 268

electrophoresis J Clin Microbiol 435483-5490 269

Gierloslashff B 1980 Pseudomonas aeruginosa IV IV Pyocine typing of strains isolated from 270

the blue fox (Alopex lagopus) mink (Mustela vison) and dog (Canis familiaris) and 271

from their environment Nord Vet Med 32147-160 272

Grundmann H Schneider C Hartung D Daschner FD Pitt TL 1995 273

Discriminatory power of three DNA-based typing techniques for Pseudomonas 274

aeruginosa J Clin Microbiol 33528-534 275

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Hammer AS Pedersen K Andersen TH Joslashrgensen JC Dietz HH 2003 Comparison 276

of Pseudomonas aeruginosa isolates from mink by serotyping and pulsed-field gel 277

electrophoresis Vet Microbiol 94237-243 278

Hariharan H Coles M Poole D Lund L Page R 2006 Update on antimicrobial 279

susceptibilities of bacterial isolates from canine and feline otitis externa Can Vet J 280

47253-255 281

Homma JY 1982 Designation of the thirteen O-group antigens of Pseudomonas aeruginosa 282

an amendment for the tentative proposal in 1976 Jpn J Exp Med 52317-320 283

Honda E Homma JY Abe C Tanamoto K Noda H Yanagawa R 1977 Effects of the 284

common protective antigen (OEP) and toxoids of protease and elastase from 285

Pseudomonas aeruginosa on protection against hemorrhagic pneumonia in mink 286

Zentralbl Bakteriol Orig A 237297-309 287

Johnson JK Arduino SM Stine OC Johnson JA Harris AD 2007 Multilocus 288

sequence typing compared to pulsed-field gel electrophoresis for molecular 289

typing of Pseudomonas aeruginosa J Clin Microbiol 453707-3712 290

Khajuria A Praharaj AK Kumar M Grover N 2013 Emergence of NDM-1 in the 291

clinical isolates of Pseudomonas aeruginosa in India J Clin Diagn Res 71328-1331 292

Knox B 1953 Pseudomonas aeruginosa som a˚rsag til enzootiske infektioner hos mink 293

Nord Vet Med 4731-760 294

Lin D Foley SL Qi Y Han J Ji C Li R Wu C Shen J Wang Y 2012 295

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated 296

from canine infections J Appl Microbiol 11316-23 297

Martino P Martino JJ Villar JA 1985 Hemorrhagic pneumonia caused by Pseudomonas 298

aeruginosa in mink in Argentina Rev Argent Microbiol 17145-148 299

Nauerby B Pedersen K Dietz HH Madsen M 2000 Comparison of Danish isolates of 300

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Salmonella enterica serovar enteritidis PT9a and PT11 from hedgehogs (Erinaceus 301

europaeus) and humans by plasmid profiling and pulsed-field gel electrophoresis J Clin 302

Microbiol 383631-3635 303

Pedersen K Hammer AS Soslashrensen CM Heuer OE 2009 Usage of antimicrobials and 304

occurrence of antimicrobial resistance among bacteria from mink Vet Microbiol 305

133115-122 306

Salomonsen CM Themudo GE Jelsbak L Molin S Hoslashiby N Hammer AS 2013 307

Typing of Pseudomonas aeruginosa from hemorrhagic pneumonia in mink (Neovison 308

vison) Vet Microbiol 163103-109 309

Shimizu T Homma JY Aoyama T Onodera T Noda H 1974 Virulence of 310

Pseudomonas aeruginosa and spontaneous spread of pseudomonas pneumonia in a mink 311

ranch Infect Immun 1016-20 312

Tam VH Chang KT Abdelraouf K Brioso CG Ameka M McCaskey LA Weston 313

JS Caeiro JP Garey KW 2010 Prevalence resistance mechanisms and 314

susceptibility of multidrug-resistant bloodstream isolates of Pseudomonas aeruginosa 315

Antimicrob Agents Chemother 541160-1164 316

Tenover FC Arbeit RD Goering RV 1997 How to select and interpret molecular strain 317

typing methods for epidemiological studies of bacterial infections a review for 318

healthcare epidemiologists Molecular Typing Working Group of the Society for 319

Healthcare Epidemiology of America Infect Control Hosp Epidemiol 18426-439 320

Wang SJ 2004 Studies on epidemiology and prevention technology of caprine 321

Pseudomonas aeruginosa disease Shandong agricultural university Masters Thesis 322

28-31 323

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Table 1 Sources serotypes and hemolysis of mink Pseudomonas aeruginosa isolates from Shandong

Isolate Serotype Hemolysis Date of collection City Source

111201 G Yes 201111 Linyi Lung

111202 G Yes 201111 Linyi Lung

111203 M Yes 201111 Linyi Lung

111204 I Yes 201111 Linyi Lung

11092615 G Yes 20119 Linyi Lung

11092616 G Yes 20119 Linyi Lung

11092617 G Yes 20119 Linyi Lung

11092618 G Yes 20119 Linyi Lung

11082616 I Yes 20118 Weihai Lung

11082623 I Yes 20118 Weihai Lung

11082639 G Yes 20118 Weihai Lung

11092304 B Yes 20119 Jinan Lung

DA G Yes 201111 Weihai Lung

DC G Yes 201111 Weihai Lung

DD G Yes 201111 Weihai Lung

DF G Yes 201111 Weihai Lung

DH G Yes 201111 Weihai Lung

DI G Yes 201111 Weihai Lung

DJ G Yes 201111 Weihai Lung

C3sl M Yes 201111 Weihai Feed

C3F9 G Yes 201111 Weihai Fecal

PA+ G Yes 201111 Weihai Lung

QT1 M Yes 201010 Weihai Lung

QT2 G Yes 201010 Weihai Lung

QT3 G Yes 201010 Weihai Lung

QT4 I Yes 201010 Weihai Lung

WD-01 G Yes 201010 Weihai Lung

JZ01 Non-typable Yes 201010 Jiaozhou Lung

02 G Yes 201010 Weihai Lung

04 G Yes 201010 Weihai Lung

Table

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Figure 1

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Figure 1