1

Paper type: Research Article 1

Subject category: Antimicrobial agents and Chemotherapy. 2

3

Clonal diversity of Acinetobacter baumannii from diabetic patients in Saudi Arabian 4

hospitals 5

Abdulrahman A. Alsultan,1 Elsayed Aboulmagd,

1,2,* Benjamin A. Evans,

3 Sebastian G. B. Amyes

4 6

7

1Department of Medical Microbiology, College of Medicine, King Faisal University, Al-Ahsa 8

31982, PO Box 400, Kingdom of Saudi Arabia 9

2Department of Microbiology, Faculty of Pharmacy, Alexandria University, El-Khartoum Square, 10

Azarita, Alexandria 21521, Egypt. 11

3Department of Life Sciences, Faculty of Science and Technology, Anglia Ruskin University, East 12

Road, Cambridge CB1 1PT, UK 13

4Department of Medical Microbiology, College of Medicine and Veterinary Medicine, University 14

of Edinburgh, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK 15

16

Running title: Clonal diversity of A. baumannii from Saudi patients 17

* Corresponding Author: Elsayed Aboulmagd, Department of Medical Microbiology, College of 18

Medicine, King Faisal University, Al-Ahsa 31982, PO Box 400, Kingdom of Saudi Arabia 19

Tel: +966-13-5800000, Fax: +966-13-5800820; Mobile: +966-541870557 20

elsayed20@hotmail.commail address: -E 21

No. of Figures: 1 22

No. of Tables: 3 23

Journal of Medical Microbiology Papers in Press. Published August 8, 2014 as doi:10.1099/jmm.0.079640-0

2

ABSTRACT 24

Carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a major health-care problem 25

causing high rates of morbidity and mortality. This study investigated the clonality of CR-AB 26

isolated from diabetic patients from different regions in Saudi Arabia as well as the relatedness of 27

the β-lactamases genes. A total of 64 non-repetitive CR-AB clinical isolates were collected from 16 28

different regions in Saudi Arabia from intensive care patients. Isolates were identified 29

phenotypically by Vitek 2 compact system and genotypically by amplification of blaOXA-51-like gene. 30

The target sequences were amplified by PCR and the clonal diversity of the isolates was explored 31

by PFGE. Resistance studies revealed that the prevalence of imipenem and meropenem resistance 32

was 92% and 96%, respectively, while the vast majority of the isolates were susceptible to 33

tigecycline and colistin. In addition, blaVIM and blaOXA-23 were the most prevalent genes in the 34

isolates under investigation while ISAba1 was the most dominant insertion sequence. PFGE results 35

showed 13 clusters; clone H was dominant comprising 20 isolates from four hospitals followed by 36

clones C and F comprising 11 isolates each from 3 and 6 hospitals, respectively. Moreover, the 37

current study signified the clonal diversity of CR-AB in Saudi Arabia and showed the ability of 38

some clones to infect patients in many different cities. 39

40

41

KEYWORDS: Acinetobacter baumannii; Carbapenem resistant, Clonal diversity; Saudi Arabia; 42

Diabetic patients 43

44

45

46

47

48

3

INTRODUCTION 49

Acinetobacter baumannii is an aerobic, non-motile, non-fermenting Gram-negative opportunistic 50

pathogen that is playing a major role in nosocomial infections of immunecompromised patients 51

(Brown and Amyes, 2006). It is considered one of the six most important multidrug-resistant 52

microorganisms in the hospitals especially in the intensive care units. Infections with this pathogen 53

are often associated with high rates of morbidity and mortality (Papp-Wallace et al., 2011). A. 54

baumannii intrinsically has low susceptibility to different antimicrobial agents (Lee et al., 2011). In 55

the last decade, many multi-drug resistant (MDR) and extensive-drug resistant A. baumannii were 56

isolated globally (Souli et al., 2008), regionally (Al-Sweih et al., 2012; Shahcheraghi et al., 2011) 57

and locally (Abdalhamid et al., 2014; Alsultan et al., 2013). Nowadays, MDR A. baumannii are 58

among the most difficult pathogens to treat (Maragakis and Perl 2008). Carbapenems represent the 59

main therapy for the serious infections caused by such pathogens (Mohajeri et al., 2013, Qi et al., 60

2008). Unfortunately, a dramatic increase of CR-AB isolates has been recorded in the recent years 61

(Alsultan et al., 2013; Shahcheraghi et al., 2011). 62

Resistance of A. baumannii to carbapenems is mainly mediated by: (i) changes in porin proteins, (ii) 63

development of efflux pumps, (iii) modification of penicillin-binding proteins, and production of 64

different types of β-lactamases (Poirel and Nordmann 2006a). According to the Ambler 65

classification (Ambler 1980), there are four classes of β-lactamases (A, B, C and D). Classes A, C 66

and D are serine type enzymes while class B is metallo-type enzymes (metallo-β-lactamases, 67

MBLs) which require zinc for their catalytic activity (Walther-Rasmussen and Høiby 2006). In A. 68

baumannii, MBLs (class B) and OXA-type carbapenemases (class D) mainly mediate resistance to 69

carbapenems and to a lesser extent class A e.g. KPC (Brown and Amyes, 2006). 70

Since the discovery of the first OXA-type carbapenemases in 1993 in A. baumannii isolate from 71

Scotland (Paton et al., 1993), many types were discovered and the number of OXA-type β- 72

lactamases exponentially increases (Lee et al., 2011). Five main groups of OXA carbapenemase are 73

involved in the resistance of A. baumannii: OXA-23-like, OXA-40-like, OXA-51-like, OXA-58- 74

4

like and OXA-143 enzymes (Brown and Amyes, 2006). The genes coding these enzymes are 75

regulated by upstream insertion sequences (IS) specifically ISAba1, ISAba2, ISAba3 and IS18 76

(Brown and Amyes, 2006; Heritier et al., 2006; Peleg et al., 2008). These insertion elements play a 77

major role in the expression of such genes (Poirel and Nordmann 2006b; Turton et al., 2006a). 78

Chromosomally located blaOXA-51-like genes are intrinsically present in all A. baumannii stains 79

(Turton et al., 2006b). In addition to OXA carbapenemases, three MBLs have been detected in A. 80

baumannii namely IMP, VIM and SIM types (Peleg et al., 2008). In Saudi Arabia, the data 81

regarding the mechanism of carbapenem resistance in A. baumannii is limited. 82

The aim of the present study was to investigate the prevalence of the genes and different insertion 83

sequences associated with carbapenem resistance in A. baumannii clinical isolates from 16 different 84

regions in Saudi Arabia. In addition, the clonal relatedness of such clinical isolates was determined. 85

86

METHODS 87

Bacterial isolates. Sixty-four non-repetitive A. baumannii clinical isolates were collected from 88

clinical specimens from diabetic patients in intensive care units. The isolates were collected from 16 89

different regions in Saudi Arabia between January and November 2012. No ethical approval was 90

required because samples were collected as a routine part of standard patient care. Conventional 91

microbiological methods were performed for preliminarily identification of the isolates and the 92

identification was confirmed by the Vitek 2 compact system (BioMerieux, Marcy L’Etoile, France) 93

according to the guidelines of the manufacturer. For molecular confirmation of the Vitek 2 compact 94

system identification, PCR was used to detect the intrinsic blaOXA-51-like gene. The primers used for 95

amplification of such gene were depicted in Table 1. 96

Antimicrobial susceptibility testing. The resistance profile of the isolates, against imipenem, 97

meropenem, tigecycline and colistin, was initially determined by Vitek 2 compact system. It was 98

subsequently confirmed by the agar diffusion assay according to Clinical and Laboratory Standards 99

Institute (CLSI) using antibiotic discs (Oxoid Ltd, UK). Minimum inhibitory concentrations (MICs) 100

5

were determined by Etest strips (AB Biodisk, Solna, Sweden) according to instructions of the 101

manufacturer. All experiments were carried out in triplicate. 102

Detection of blaOXA-23 and blaOXA-40 genes. Polymerase chain reaction (PCR) was used to amplify 103

the genes encoding OXA-type carbapenemases (blaOXA-23, blaOXA-40) and MBLs (blaVIM, blaSIM, 104

blaGIM, blaIMP and blaSPM) as previously described (Ellington et al., 2007; Qi et al., 2008; Poirel and 105

Nordmann 2006b; Woodford et al., 2006) using the primers depicted in Table 1. 106

Molecular typing by PFGE. Sixty four A. baumannii clinical isolates were typed by PFGE 107

analysis as previously described (Miranda et al., 1996). DNA obtained from bacteria was digested 108

using the Apa1 restriction endonuclease (Promega, Southampton, UK), and DNA fragments were 109

separated on 1% agarose gel in 0.5× TBE buffer using the CHEF-RDII apparatus (Bio-Rad, UK). 110

The conditions used were the following: pulse time 5-35s at field strength of 6v/cm for 24h at 37C. 111

The gel was stained by ethidium bromide and then the digital images were captured by Gel doc2000 112

(Bio-Rad, UK). All isolates were analyzed using Bionumerics software version 6.5. Isolates that 113

clustered together with a similarity of >85% were considered to belong to the same PFGE type. 114

115

RESULTS 116

Clinical isolates and antimicrobial susceptibility 117

Sixty-four non-repetitive A. baumannii clinical isolates (coded DM001 to DM064) were collected 118

from 16 different regions in Saudi Arabia. The isolates were cultured from different clinical 119

specimens of diabetic patients admitted to ICUs. Thirty-four patients (53%) were male while the 120

female patients represented 47% (30/64). Most isolates were collected from respiratory tract clinical 121

specimens (41 out of 64, 64%) while the rest of the isolates were obtained from infected blood, 122

urine, abdomen and skin. Vitek II compact system was used to identify the isolates and the 123

identification was confirmed after amplification of the intrinsic blaOXA-51-like gene by PCR. 124

6

All isolates showed resistance to at least one of the two tested carbapenems (imipenem and 125

meropenem) as shown in Fig. 1. No significant difference between the prevalence of imipenem (59 126

out of 64, 92%) and meropenem (62 out of 64, 96%) resistance among the tested isolates as shown 127

in Table 2. On the other hand, the majority of the isolates showed susceptibility to tigecycline and 128

colistin, 97% (Table 2). 129

Prevalence of β-lactamases and insertion sequences 130

The prevalence of the different genes coding two OXA carbapenemases (OXA-23 and OXA-40), 131

five MBLs (VIM, IMP, SPM, SIM and GIM) and the different insertion sequences (ISs) was 132

calculated after amplification of the target genes by PCR and the results were depicted in Table 3. 133

Thirty-four (53.1%) and 19 (29.7%) isolates harbored blaOXA-23 and blaOXA-40, respectively. Only 134

two MBLs were detected in the isolates where blaVIM was significantly more prevalent than blaSPM, 135

59/64 (92.2%) and 18/64 (28.1%), respectively. On the other hand, blaIMP, blaSIM and blaGIM were 136

not detected in any of the tested isolates (Table 3). Although IS18 was absent from all the tested 137

isolates, ISAba1, ISAba2 and ISAba3 were amplified from 58 (90.6%), 6 (9.4%) and 13 (20.3%) of 138

the tested isolates, respectively (Table 3). 139

PFGE genotyping 140

PFGE was applied to study the clonal diversity and relatedness of the tested isolates. The 141

discrimination power of PFGE technique was expressed by dice coefficient with BioNumerics 142

software version 6.5. The clustering of 13 PFGE groups, A to M, detailing the ID number (isolate 143

code), sex, locations, transferable OXA β-lactamase (OXA-23, OXA-40), insertion sequences 144

(ISAba1, ISAba2, ISAba3 and IS18) and MBLs (VIM, SIM, GIM, IMP and SPM) were shown in 145

Fig. 1. All detected PFGE patterns showed a genetic similarity coefficient ranging from 70% to 146

100%; however isolates that clustered together with a similarity greater than 85% were considered 147

to comprise the same PFGE type. Clone C and F each comprised 11 isolates from four and three 148

cities respectively, whereas and H comprised 20 isolates from six cities (Fig. 1). In addition, nine 149

7

and five different clones were detected in 34 and 6 isolates from Riyadh and Almadinah, 150

respectively. 151

152

DISCUSSION 153

The emergence and global distribution of CR-AB represent a major problem in the health care 154

settings, especially ICUs. A. baumannii is a notorious opportunistic pathogen mainly associated 155

with hospital-acquired infections (Wang et al., 2013). This pathogen causes serious hospital 156

acquired infections associated with high mortality rate particularly in immunocompromised 157

patients. Moreover, very limited therapeutic options (e.g. colistin and tigecycline) are available for 158

treatment of infection cause by such pathogens (Bassetti et al., 2011). 159

The recent worldwide emergence of carbapenem resistance in Gram negatives has mainly 160

manifested by the infiltration of different types of β-lactamases. Carbapenems were the drug of 161

choice for treating infections caused by MDR A. baumannii, however, resistance to such 162

antimicrobial agents is now a common occurrence and pan-drug resistant strains are beginning to 163

emerge (Brown and Amyes, 2006). In A. baumannii, the main carbapenem hydrolyzing β- 164

lactamases are OXA-type carbapenemases (Ambler class D β-lactamases) and MBLs (class B β- 165

lactamases) (Poirel et al., 2012). 166

In the present study, 64 A. baumannii clinical isolates (based on the presence of blaOXA-51-like gene) 167

were collected from tertiary care hospitals located in 16 different cities in Saudi Arabia. These cities 168

were distributed in many different provinces separated by large geographic distances. The majority 169

of the isolates were recovered from respiratory tract secretions. The vast majority of the isolates 170

were susceptible to colistin and tigecycline (97%). Higher tigecycline resistance rate (9.7%) was 171

recently recorded in Riyadh region (Baadani et al., 2013) while susceptibility to colistin in the 172

current study is in accordance with previously published reports (Al-Agamy et al., 2014; Lambiase 173

et al., 2012; Oteo et al., 2007). On the other hand, Shahcheraghi and his co-workers reported that 174

12% of A. baumannii isolates in Iran were resistant to colistin (Shahcheraghi et al., 2011) although 175

8

in another Iranian study no colistin resistance was observed in 104 clinical isolates (Mohajeri et al., 176

2013). Although many colistin and tigecycline resistant A. baumannii pathogens have recently been 177

isolated (Agodi et al., 2014, Bahador et al., 2013), these two antimicrobial agents remain the drug 178

of choice for combating infections caused by CR-AB. 179

The prevalence of OXA-23 in the current study was 53.1% which is comparable with recently 180

published result from Egypt (50%) (Al-Agamy et al., 2014) and India (47.9%) (Karunasagar et al., 181

2011). Higher prevalence of OXA-23 was detected in CR-AB isolates from Riyadh (100%) (Aly et 182

al., 2014), the Eastern region of Saudi Arabia (80.4%) (Abdalhamid et al., 2014) and Iran [84% 183

(Shahcheraghi et al., 2011) and 77.9% (Mohajeri et al., 2013)]. In contrast, only two isolates carried 184

blaOXA-23 out of 40 (5%) isolates collected from Kuwait (Al-Sweih et al., 2012) while only one 185

isolate out of 92 (1.1%) isolated from Taiwan harbored such gene (Lu et al., 2009). On the other 186

hand, blaOXA-40 was amplified from 29.7% of our isolates. This carbapenemase was not detected in 187

any of the 253 tested isolates from Riyadh (Aly et al., 2014). While blaOXA-40 was the most 188

prevalent acquired gene (57.6%) in 59 isolates from Spain, none of those isolates harbored blaOXA-23 189

(Villalón et al., 2013). In addition, 7.5%, 19.2% and 22.9% of isolates from Egypt (Al-Agamy et 190

al., 2014), Iran (Mohajeri et al., 2013) and India (Karunasagar et al., 2011), respectively, contained 191

the OXA-40 β-lactamase. Co-existence of both blaOXA-23 and blaOXA-40 was not detected in any of 192

our isolates. In contrast, 45% and 16.4% CRAB isolates collected from Egypt (Al-Agamy et al., 193

2014) and Iran (Mohajeri et al., 2013), respectively, co-produced both the OXA-23 and OXA-40 β- 194

lactamases. 195

Beside OXA carbapenemases, MBLs were detected in many of the isolates. VIM and SPM β- 196

lactamases were the only two MBLs detected where their prevalence was 92.2% (59/64) and 28.1% 197

(18/64), respectively, while the other three MBLs (IMP, GIM and SIM) were absent. Lower 198

prevalence of MBLs (6%) has been recorded in Iran (Shahcheraghi et al., 2011) where only blaSPM 199

was detected while blaVIM was completely absent. In addition, 46 CRAB clinical isolates from the 200

Eastern region of Saudi Arabia harbored neither blaVIM nor blaIMP (Abdalhamid et al., 2014). 201

9

Moreover, MBL encoding genes were not amplified from any of 40 clinical isolates from Egypt 202

(Al-Agamy et al., 2014). In contrast, 72.5% (29/40) CRAB isolates from Kuwait carried bla genes 203

encoding VIM and IMP MBLs (Al-Sweih et al., 2012). 204

Our results revealed that ISAba1 was the most prominent (90.6%) insertion element followed by 205

ISAba3 (20.3%), while ISAba2 was detected in only 6 isolates out of 64 (9.4%). The prevalence of 206

ISAba1 is comparable with that recorded in 59 isolates from Spain (93.2%) (Villalón et al., 2013). 207

Lower prevalence was recorded in Taiwan (36%) (Lu et al., 2009) and India (33%) (Karunasagar et 208

al., 2011). Presence of different insertion sequences renders the A. baumannii resistant to 209

carbapenems (Lee et al., 2011). Such insertion sequences located in the proximity of genes coding 210

different OXA-types carbapenemases and involved in their overexpression (Corvec et al., 2003). 211

In the current work, the genetic similarity of PFGE types was very high (89 to 100%). The clonal 212

diversity revealed two types of epidemic clones: monoclonal and polyclonal. The monoclonal 213

model showed the most common clones appeared in 12 out of 16 cities. These monoclonal 214

outbreaks were caused by either one or more of epidemic PFGE type. The polyclonal model has 215

affected four cities (Riyadh, Almadinah, Tabok and Kamis-Mosait). Riyadh is the capital city of 216

Saudi Arabia, and patients possessed nine out of the 13 different clones while Almadinah, the 217

capital of Almadinah province, had 5 different clones. Both cities might cause explosive outbreaks 218

at different time. Clone F has been detected in hospitals of Riyadh, Almadinah, Tabok, Abha, 219

Alrass and Alkafji. This finding suggests transmission of clones not only from one hospital to 220

another but also between distant health regions. The promiscuity of A. baumannii transmission has 221

been recognized (Coelho et al., 2006; Dijkshoorn et al., 1996; Manikal et al., 2000) suggesting 222

reappearance of certain clones within these hospitals reflects endemic persistence of this pathogen 223

in diabetic patients, hospital and environments which may represent a risk factor for future 224

outbreaks. 225

In conclusion, the result of the current study revealed that OXA-23 and VIM were the most 226

common β-lactamases conferring carbapenem resistance to A. baumannii. In addition, ISAba1 was 227

10

the most prevalent insertion sequence. Moreover, the current study not only signifies the clonal 228

diversity of CR-AB in Saudi Arabia but also the ability of some clones to infect patients in many 229

different cities. 230

ACKNOWLEDGMENT 231

The authors are grateful for financial support by King Abdulaziz City for Science and Technology 232

(KACST). 233

234

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Acinetobacter baumannii. FEMS Microbiol Lett 258, 72-77. 339

Turton, J. F., Woodford, N., Glover, J., Yarde, S., Kaufmann, M. E. & Pitt, T. L. (2006b). 340

Identification of Acinetobacter baumannii by detection of the blaOXA-51-like carbapenemase gene 341

intrinsic to this species. J Clin Microbiol 44, 2974-2976. 342

Villalón, P., Valdezate, S., Medina-Pascual, M. J., Carrasco, G., Vindel, A. & Saez-Nieto, J. A. 343

(2013). Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing 344

oxacillinase and metallo-β-lactamase genes, and of common insertion sequences, in epidemic 345

clones of Acinetobacter baumannii from Spain. J Antimicrob Chemother 68, 550-553. 346

Walther-Rasmussen, J. & Høiby, N. 2006. OXA-type carbapenemases. J Antimicrob Chemother 347

57, 373-383. 348

Wang, X., Qiao, F., Yu, R., Gao, Y. & Zong, Z. (2013). Clonal diversity of Acinetobacter 349

baumannii clinical isolates revealed by a snapshot study. BMC Microbiol 13, 234. 350

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Woodford, N., Ellington, M. J., Coelho, J. M., Turton, J. F., Ward, M. F., Brown, S., Amyes, 351

S. G. & Livermore, D. M. (2006). Multiplex PCR for genes encoding prevalent OXA 352

carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 27, 351-353. 353

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371

372

16

Table 1. Nucleotide sequence of primers used in this study 373

Primer name Nucleotide Sequence (5`- 3`) Reference

OXA-51-F TAATGCTTTGATCGGCCTTG Woodford et al., 2006

Woodford et al., 2006 OXA-51-R TGGATTGCACTTCATCTTGG

OXA-23-F GATCGGATTGGAGAACCAGA Qi et al., 2008

Qi et al., 2008 OXA-23-R ATTCTTGACCGCATTTCCAT

OXA-40-F GGTTGTTGGCCCCCTTAAA Qi et al., 2008

Qi et al., 2008 OXA-40-R AGTTGAGCGAAAAGGGGATT

ISAba1-F GTGCTTTGCGCTCATCATGC Poirel and Nordmann 2006b

Poirel and Nordmann 2006b ISAba1-R CATGTAAACCAATGCTCACC

ISAba2-F AATCCGAGATAGAGCGGTTC Poirel and Nordmann 2006b

Poirel and Nordmann 2006b ISAba2-R TGACACATAACCTAGTGCAC

ISAba3-F CAATCAAATGTCCAACCTGC Poirel and Nordmann 2006b

Poirel and Nordmann 2006b ISAba3-R CGTTTACCCCAAACATAAGC

IS18-F CACCCAACTTTCTCAAGATG Poirel and Nordmann 2006b

Poirel and Nordmann 2006b IS18-R ACCAGCCATAACTTCACTCG

IMP-F GGAATAGAGTGGCTTAAYTCTC Ellington et al., 2007

Ellington et al., 2007 IMP-R CCAAACYACTASGTTATCT

VIM-F GATGGTGTTTGGTCGCATA Ellington et al., 2007

Ellington et al., 2007 VIM-R CGAATGCGCAGCACCAG

GIM-F TCGACACACCTTGGTCTGAA Ellington et al., 2007

Ellington et al., 2007 GIM-R AACTTCCAACTTTGCCATGC

SPM-F AAAATCTGGGTACGCAAACG Ellington et al., 2007

Ellington et al., 2007 SPM-R ACATTATCCGCTGGAACAGG

SIM-F TACAAGGGATTCGGCATCG Ellington et al., 2007

Ellington et al., 2007 SIM-R TAATGGCCTGTTCCCATGTG

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375

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377

378

17

Table 2. Phenotypic characteristics of sixty-four A. baumannii clinical isolates 379

Antimicrobial agents

Number of isolates (%)

Sensitive (S) Intermediate (I) Resistant (R)

Imipenem 3 (4.7) 2 (3.1) 59 (92.2)

Meropenem 0 (0) 2 (3.1) 62 (96.9)

Tigecycline 62 (96.9) 2 (3.1) 0

Colistin 62 (96.9) 0 2 (3.1)

380

381

382

383

384

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387

388

389

390

391

392

393

394

18

Table 3. Genotypic characteristics of sixty-four A. baumannii clinical isolates 395

Characteristic Number of isolates (%)

blaOXA-23 34 (53.1)

blaOXA-40 19 (29.7)

Co existence of both blaOXA-40 and blaOXA-23 0 (0)

Absence of both blaOXA-40 and blaOXA-23 11 (17.2)

blaVIM 59 (92.2)

blaSPM 18 (28.1)

Co existence of both blaVIM and blaSPM 17 (26.6)

blaIMP 0 (0)

blaGIM 0 (0)

BlaSIM 0 (0)

ISAba1 58 (90.6)

ISAba2 6 (9.4)

ISAba3 13 (20.3)

IS18 0 (0)

Co existence of both ISAba1 and ISAba2 5 (7.8)

Co existence of both ISAba1 and ISAba3 11 (17.2)

396

397

398

399

19

400

401 402

100 90 80 70

DM003 DM015 DM012 DM013 DM045 DM048 DM063 DM028 DM029 DM030 DM032 DM037 DM057 DM047 DM055 DM021 DM064 DM031 DM034 DM004 DM044 DM060 DM001 DM002 DM040 DM039 DM041 DM026 DM061 DM052 DM058 DM042 DM062 DM053 DM006 DM007 DM008 DM009 DM010 DM011 DM043 DM059 DM033 DM035 DM036 DM054 DM056 DM022 DM023 DM024 DM025 DM046 DM049 DM051 DM027 DM050 DM005 DM038 DM014 DM016 DM018 DM019 DM020 DM017

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M F M F M M M F F F F F F F M M M M M M F F M M M F F M M F M M F M F F F F F F M M M M M M M F F F F M M M F F M F F F M M M M

RIYADH RIYADH RIYADH BREDAH ALMADINAH RIYADH RIYADH TABOK TABOK TABOK TABOK RIYADH RIYADH RIYADH RIYADH ANAK HAIL ALMADINAH RIYADH RIYADH ALDAWAD. ALDAWAD. ALMADINAH ALMADINAH RIYADH ABHA RIYADH RIYADH RIYADH ALRASS ALKAFJI TABOK RIYADH RIYADH RIYADH RIYADH RIYADH RIYADH RIYADH RIYADH JAZAN RIYADH ASEER ASEER ASEER RIYADH RIYADH RIYADH RIYADH RIYADH RIYADH ASEER ASEER ALMADINAH SHAGRA ALMADINAH KAMIS M ALAHSA KAMIS M ALQASEM RIYADH RIYADH RIYADH RIYADH

- + + + + + + - - - - + + + + + - + + + + + + + + + + + + + + + - + - - - - - - - - - - - - + - - - - - - - + - - - + - + + + +

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + + + + + + + + + - + + + + + + + - - - - - - - - - -

- + + + + + + + + + + + - + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + - + - + + + + + + + +

+ - + - - - - - - - - - - - - - - - - + + - + + - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - + + - - - - - - - + - - - - - - + - - - - - - - - - - - + - - - - - - + - + + + + - - - - - + + + - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- + + + + + + + + + + + + + + - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + - + + - + +

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

+ - - - + + + - - - - - - - + - + + - - + + + + - + - + + - - - + - - - - - - - - + - - - - - - - - - + + - - - - - - - - - - -

R R R R R R R R R R R R R R S R R R R R R R R R R R R R R R R R I R R R R R R R R R R R R R R R R R R R R I R S R S R R R R R R

R R R R R R R R R R R R R R I R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R I R R R R R R R R

S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S I S S S S S I S S S S S S S S S S S S S S S S S S S S S S S S S

S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S R S S R S S S S S S S S S S S S S S S S S S S S S S S S

Genetic similarity index (%)

OXA ISAba IS Metallo-β-lactamase ID Sex Location 23 40 1 2 3 18 VIM SIM GIM IMP SPM IPM MEM TGC CST CLONES

Clone D

Clone F

CloneA1

Clone C

Clone E

Clone H

Clone

I Clone J

Clone M

Clone K

Clone G

Clone B

Clone L

F1

F2

H2

H1

C1

C2

C3

CloneA2

C4

Clone C

D1

D2

20

403

Fig. 1. Pulsed field gel electrophoresis dendrogram of 64 A. baumannii clinical isolates. The 404

broken line corresponds to the cutoff level (85%) used to define single PFGE clones. Dotted squares 405

mark the boundaries of clusters B to F, H to J and M. Abbreviations: DM, diabetes mellitus; IPM, 406

imipenem; MEM, meropenem; TGC, tigecycline; CST, colistin; R, resistance; S, susceptible; I, 407

intermediate. 408

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