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[Full Text] Cayô et al, 2011 1 Analysis of Genes Encoding for Penicillin-Binding Proteins in Clinical Isolates of 1 Acinetobacter baumannii 2 3 Rodrigo Cayô 1* , María-Cruz Rodríguez , Paula Espinal 2 , Felipe Fernández-Cuenca 3 , 4 Alain A. Ocampo-Sosa 1 , Álvaro Pascual 3 , Juan A. Ayala 4 , Jordi Vila 2 and Luis 5 Martínez-Martínez 1,5 . 6 7 Short running title: Analysis of Penicillin-Binding Proteins in A. baumannii. 8 9 1 Service of Microbiology, University Hospital Marqués de Valdecilla - IFIMAV, 10 Santander, Spain; 11 2 Service of Microbiology, Hospital Clinic, School of Medicine, University of 12 Barcelona, Barcelona, Spain; 13 3 Service of Microbiology, University Hospital Virgen Macarena, Seville, Spain; 14 4 Centro de Biología Molecular Severo Ochoa - CSIC-UAM, Campus de Cantoblanco, 15 Madrid, Spain. 16 5 Department of Molecular Biology, School of Medicine, University of Cantabria, 17 Santander, Spain. 18 § Current address: Instituto de Biomedicina y Biotecnología de Cantabria, Santander, 19 Spain. 20 21 *Corresponding author. 22 Current Address: Laboratorio Especial de Microbiologia Clínica - LEMC/ALERTA, 23 Universidade Federal de São Paulo - UNIFESP, Rua Leandro Dupret, 188, Vila 24 Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Antimicrob. Agents Chemother. doi:10.1128/AAC.00459-11 AAC Accepts, published online ahead of print on 26 September 2011 on January 30, 2018 by guest http://aac.asm.org/ Downloaded from

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Page 1: 1 Analysis of Genes Encoding for Penicillin-Binding Proteins in

[Full Text] Cayô et al, 2011

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Analysis of Genes Encoding for Penicillin-Binding Proteins in Clinical Isolates of 1

Acinetobacter baumannii 2

3

Rodrigo Cayô1*, María-Cruz Rodríguez1§, Paula Espinal2, Felipe Fernández-Cuenca3, 4

Alain A. Ocampo-Sosa1, Álvaro Pascual3, Juan A. Ayala4, Jordi Vila2 and Luis 5

Martínez-Martínez1,5. 6

7

Short running title: Analysis of Penicillin-Binding Proteins in A. baumannii. 8

9

1Service of Microbiology, University Hospital Marqués de Valdecilla - IFIMAV, 10

Santander, Spain; 11

2Service of Microbiology, Hospital Clinic, School of Medicine, University of 12

Barcelona, Barcelona, Spain; 13

3Service of Microbiology, University Hospital Virgen Macarena, Seville, Spain; 14

4Centro de Biología Molecular Severo Ochoa - CSIC-UAM, Campus de Cantoblanco, 15

Madrid, Spain. 16

5Department of Molecular Biology, School of Medicine, University of Cantabria, 17

Santander, Spain. 18

§Current address: Instituto de Biomedicina y Biotecnología de Cantabria, Santander, 19

Spain. 20

21

*Corresponding author. 22

Current Address: Laboratorio Especial de Microbiologia Clínica - LEMC/ALERTA, 23

Universidade Federal de São Paulo - UNIFESP, Rua Leandro Dupret, 188, Vila 24

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00459-11 AAC Accepts, published online ahead of print on 26 September 2011

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Clementino, 04025-010, São Paulo - SP, Brazil. Tel.: +55 11 50812965. Fax.: +55 11 25

50812965. E-mail: [email protected]. 26

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ABSTRACT 27

There is limited information on the role of penicillin-binding proteins (PBPs) in the 28

resistance of Acinetobacter baumannii to β-lactams. This study presents an analysis of 29

the allelic variations of PBP genes in A. baumannii isolates. Twenty six A. baumannii 30

clinical isolates (susceptible or resistant to carbapenems) from three teaching hospitals 31

in Spain were included. Antimicrobial susceptibility profile, clonal pattern and genomic 32

species identification were also evaluated. Based on the six complete genomes of A. 33

baumannii, the PBP genes were identified and primers were designed for each gene. 34

The nucleotide sequences of the identified genes encoding for PBPs and the 35

corresponding amino acid sequences were compared with those of the ATCC 17978 36

strain. Seven PBP and one monofunctional transglycosylase genes were identified in the 37

six genomes, encoding for (i) four high molecular mass proteins [two of class A: ponA 38

(PBP1a) and mrcB (PBP1b), and two of class B: pbpA/mrdA (PBP2) and ftsI (PBP3)], 39

(ii) three low molecular mass proteins [two of Type-5: dacC (PBP5/6) and dacD 40

(PBP6b)], one Type-7 [pbpG (PBP7/8)] and (iii) a monofunctional enzyme [mtgA 41

(MtgA)]. Hotspot mutation regions were observed, although most of the allelic changes 42

found translated into silent mutations. The amino acid consensus sequence of the PBP 43

genes in the genomes and the clinical isolates were highly conserved. The changes in 44

amino acid sequence were associated to concrete clonal patterns, but were not directly 45

related to susceptibility or resistance to β-lactams. An insertion sequence disrupting the 46

gene encoding the PBP6b was identified in an endemic carbapenem-resistant clone in 47

one of the participant hospitals. 48

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INTRODUCTION 49

Acinetobacter baumannii is an opportunistic nosocomial pathogen responsible for a 50

variety of serious infections, especially in intensive care units (ICU) (23,27). Its ability 51

to survive on dry surfaces (5,20,41) and to acquire antimicrobial resistance, as well as 52

being suited for genetic exchange have contributed to the success and longevity of this 53

microorganism to cause epidemic spread outbreaks (18). Carbapenems are currently one 54

of the few options for treatment of infections caused by multi-drug resistant A. 55

baumannii (14). However, since the early 1990s, outbreaks caused by carbapenem-56

resistant A. baumannii isolates have increased worldwide (4,15), becoming a significant 57

public health concern (40). 58

The mechanisms underlying resistance to carbapenems in A. baumannii include: (I) 59

production of beta-lactamases, particularly acquired carbapenem-hydrolyzing class D β-60

lactamases (CHDLs) (6,31), metallo-β-lactamases (30) and in rare cases, class A 61

carbapenemases (32); (II) outer membrane impermeability associated with the loss or 62

decreased expression of porins (25) and, probably, (III) overproduction of efflux pumps 63

(19). However, there is limited information on the role of penicillin-binding proteins 64

(PBPs) on this phenotype in A. baumannii (11,12,26). 65

PBPs are a family of enzymes that share a common evolutionary origin. These enzymes 66

catalyze the synthesis of peptidoglycan, the primary component of the bacterial cell 67

wall, and are also associated with cell morphogenesis and cell division complex (33). 68

PBPs have been classified into two groups, the high molecular mass (HMM) and the 69

low molecular mass (LMM) PBPs, according to their apparent molecular weight on 70

sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), amino acid sequences and 71

enzymatic and cellular functions (13,16,21,33). The HMM PBPs can be divided in class 72

A (transpeptidase/glycosyltransferase activities) (2,3) or class B PBPs (transpeptidase 73

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activity, elongase activity or divisome) (9,29), depending on the structure of their N-74

terminal domain (33). The LMM PBPs or class C PBPs, are DD-carboxypeptidades 75

and/or endopeptidases involved in cell separation, peptidoglycan maturation or 76

recycling (13). The monofunctional enzymes (MGTs) is a group of enzymes present in 77

some bacteria, with a single glycosyltransferase domain similar to those of class A 78

PBPs, and their function is still unknown (35). 79

This study aimed to determine the nucleotide sequence of the genes encoding for PBPs 80

in A. baumannii and to analyze their allelic variations in isolates susceptible or resistant 81

to β-lactams. (This work was presented in part as an oral presentation at the 8th 82

International Symposium on the Biology of Acinetobacter in Rome, 2010). 83

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MATERIALS AND METHODS 84

Bacterial isolates. 85

A total of 26 nonduplicate A. baumannii clinical isolates, presenting different 86

susceptibility profiles to carbapenems were collected in three teaching hospitals in 87

Spain: University Hospital Marqués de Valdecilla, Santander (n=12), Hospital Clínic, 88

Barcelona (n=12), and University Hospital Virgen Macarena, Seville (n=2), as shown in 89

Table 1. The two isolates from the third hospital were previously described (11). These 90

isolates were representative of the most prevalent clones in each institution. 91

Presumptive identification of the isolates as A. baumannii was done by amplifying the 92

complete open reading frame of blaOXA-51-like gene, which is considered chromosomally 93

intrinsic to A. baumannii (31), using primers pairs OXA-69A and OXA-69B as 94

previously described (17). Both primers were also used to detect the presence of ISAba1 95

upstream of the blaOXA-51-like (17). The amplified ribosomal DNA restriction analysis 96

(ARDRA) method, using the enzymes CfoI, AluI, MboI, RsaI, MspI, was carried out as 97

described before (39), to confirm the genomic species identification of A. baumannii. 98

The reference strain A. baumannii RUH-134 (10) was included as control for both 99

genomic identification and PCR amplification of PBP genes. 100

101

Antimicrobial susceptibility testing 102

Minimum inhibitory concentrations (MICs) of tigecycline and colistin were determined 103

by broth microdilution in the three participating centers, according to the Clinical 104

Laboratory Standard Institute (CLSI) guidelines (7). MICs of imipenem, meropenem, 105

cefepime, ceftazidime, ceftriaxone, cefotaxime, aztreonam, amikacin, gentamicin, 106

minocycline and ciprofloxacin were also determined by microdilution, for the isolates 107

from the Hospital Clínic and University Hospital Virgen Macarena, while they were 108

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determined with Etest strips, according to the manufacturer’s (AB bioMérieux, Solna, 109

Sweden) recommendations, for the isolates from the University Hospital Marqués de 110

Valdecilla. Results for tigecycline were interpreted according to the US Food and Drug 111

Administration (FDA) breakpoints for Enterobacteriaceae (susceptible ≤ 2 µg/mL, 112

intermediated 4 µg/mL, resistant ≥ 8 µg/mL) and for the other antimicrobial agents 113

tested according to the CLSI breakpoints (8). Pseudomonas aeruginosa ATCC 27853 114

and Escherichia coli ATCC 25922 were used as quality control strains. 115

116

Molecular typing by pulsed field gel electrophoresis (PFGE) 117

The clonal relationship of the A. baumannii isolates was determined by PFGE, using the 118

restriction enzyme ApaI (Roche Diagnostics, Indianapolis, IN, USA), as described 119

elsewhere (34). The restriction fragments were separated on a 1% (w/v) agarose gels in 120

0.5% TBE buffer in a CHEF-DR III Mapper electrophoresis system (Bio-Rad 121

Laboratories, Richmond, CA, USA) for 19 h at 14ºC using a pulse ramping rate 122

changing from 5 to 20 s at 6 V/cm. DNA fingerprints were interpreted as recommended 123

by Tenover et al. (37). The reference strains A. baumannii RUH-875 and RUH-134 124

(10), representatives of major European clones I and II, respectively, were also used as 125

comparators. 126

127

Identification of PBP genes 128

The genes encoding for PBPs were identified based on the information of the six 129

complete genomes of A. baumannii deposited in GenBank when this study was started. 130

The following organisms were considered: A. baumannii strain AB0057 (accession no. 131

NC_011586), A. baumannii strain ATCC 17978 (accession no. NC_009085), A. 132

baumannii strain SDF (accession no. NC_010400), A. baumannii strain AYE (accession 133

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no. NC_010410), A. baumannii strain ACICU (accession no. NC_010611) and A. 134

baumannii strain AB307-0294 (accession no. NC_011595). Consensus sequences were 135

obtained for each PBP gene identified and a series of primers for PCR amplification and 136

sequencing were designed, as listed in Table 1S. The genomes of A. baumannii 137

deposited in the GenBank after the beginning of these study, were also considered for 138

comparison and analysis of the PBP genes. These genomes include: A. baumannii strain 139

AB056 (accession no. NZ_ADGZ00000000), A. baumannii strain AB058 (accession no. 140

NZ_ADHA00000000), A. baumannii strain AB059 (accession no. 141

NZ_ADHB00000000), A. baumannii strain AB900 (accession no. 142

NZ_ABXK00000000), and A. baumannii strain ATCC 19606 (accession no. 143

NZ_ACQB00000000). 144

145

Analysis of the PBP genes in A. baumannii clinical isolates 146

Genomic DNA from clinical isolates was extracted with InstaGeneTM Matrix (Bio-Rad 147

Laboratories, Hercules, CA, USA), according to manufacturer’s recommendations. PBP 148

genes were amplified by PCR using the following conditions: 95ºC for 5 min, followed 149

by 35 cycles of 95ºC for 30 s, 55ºC for 30 s and 72ºC for 1 to 3 min, according to the 150

amplicon size, followed by 72ºC for 7 min. PCR products were analyzed on a 1 % (w/v) 151

agarose gels stained with ethidium bromide. PCR products were purified by using a 152

High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany). 153

Bidirectional DNA sequencing was performed by Macrogen Inc. (Seoul, South Korea). 154

Each PBP gene was named and classified based on the homologies obtained with others 155

PBP sequences from different microorganisms, deposited in the GenBank, as well as on 156

the recognition of the conserved motifs. All PBP sequences were compared with those 157

of A. baumannii ATCC 17978 (1). 158

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Nucleotide sequence accession numbers 159

The nucleotide sequences of the PBP genes of A. baumannii clinical isolates have been 160

deposited in the GenBank nucleotide sequence database and were assigned the 161

accession numbers, according to each PBP/MGT gene and strain, as follow: ponA 162

(JF746077 to JF746102), mrcB (JF746103 to JF746128), pbpA (JF745973 to 163

JF745998), ftsI (JF745999 to JF746024), dacC (JF746025 to JF746050), dacD 164

(JF746051 to JF746074), pbpG (JF746129 to JF746154) and mtgA (JF745947 to 165

JF745972). 166

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RESULTS 167

PBP genes in A. baumannii 168

Seven PBP and one MTG genes were identified in the six A. baumannii genomes 169

analyzed in this study, which are described in Table 2. Four HMM PBPs were found: 170

PBP1a (ponA), PBP1b (mrcB), PBP2 (pbpA or mrdA) and PBP3 (ftsI). According to 171

their N-terminal domain, they were classified as Class A (PBP1a and PBP1b) or Class B 172

(PBP2 and PBP3). Sequence alignment revealed the five conserved motifs 173

(EDxxFxxHxG, GxSTxx(M/Q)QxxK, RKxxE, KxxIxxYxN and RxxxxL) of the 174

glycosyltranferase N-terminal domain in both HMM class A PBPs (Figure 1A). In the 175

C-terminal penicillin-biding (PB) domain, with transpeptidase activity, the residue 176

following motif 3 [K(T/S)GT] was a Threonine in Class A PBPs (KTGTT for PBP1a 177

and KSGTT for PBP1a) and an Alanine in class B PBPs (KTGTA), as expected (Figure 178

1B). The HMM of class A, PBP1a and PBP1b, are the major transglycosylases-179

transpeptidases in A. baumannii, and they are probably involved in the elongation of 180

uncross-linked glycan chains of peptidoglycan. 181

The presence of a residue of Aspartic acid at the third position of motif 2 (SxD) found 182

in the active site of the PBP2 (pbpA), and one residue of Asparagine at the same 183

position in PBP3, confirmed the classification of these PBPs in subclasses B2 and B3, 184

respectively (Figure 1C). Both PBP2 and PBP3 are monofunctional transpeptidases. 185

PBP2 is as member of subclass B2, a group of proteins involved in cell elongation. 186

PBP3, as a member of the subclass B3, is probably associated with the cell division. 187

The fstI gene was localized in the A. baumannii genomes in an operon with mraW, 188

mraY, ftsL, murE and murF genes, which are associated with the divisome. The MtgA 189

(mgtA) is a monofunctional enzyme (MGT) that presents a glycosyltranferase domain 190

similar to those of PBP1a and PBP1b. 191

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Three LMM PBPs have been found in A. baumannii: PBP5/6 (dacC), PBP6b (dacD) 192

and PBP7/8 (pbpG) and are associated with cell separation and maturation or recycling 193

peptidoglycan. The analysis of the genes encoding for PBP5 and PBP6 in A. baumannii 194

genomes showed that they are identical, at nucleotide and amino acid levels. Thus, this 195

PBP will be referred as PBP5/6 (gene dacC). This PBP together with PBP6b (dacD) 196

were classified as class C Type-5 PBPs, and both are presumably D-ala-D-ala-197

carboxypeptidases. The PBP7/8 (pbpG) is a class C Type-7 PBP with putative 198

endopeptidase activity. No class C Type 4 PBP genes seems to be present in A. 199

baumannii. 200

201

Hotspot mutations in PBP genes and ββββ-lactams resistance 202

The 26 clinical isolates were identified as A. baumannii by ARDRA and all isolates 203

carried a blaOXA-51 allele (Table 1). PFGE profile analysis revealed that the 26 isolates 204

were categorized into 11 different clones. All A. baumannii isolates were susceptible to 205

colistin and tigecycline, with MICs ranging from 0.25 to 1 µg/ml and 0.5 to 1 µg/ml, 206

respectively. Minocycline also showed a good coverage against the A. baumannii 207

isolates tested (73.1% of susceptibility; MIC50, 2 µg/ml). High resistant rates were 208

observed for third-generation cephalosporins (MIC50, ≥ 32 µg/ml), cefepime (MIC50, ≥ 209

32 µg/ml) and aztreonam (MIC50, > 16 µg/ml). MICs of imipenem and meropenem 210

ranged from 0.25 to > 32 µg/ml, and for 53% of the isolates were > 32 µg/ml for both 211

antimicrobial agents. The majority of the isolates were resistant to ciprofloxacin (MIC50, 212

> 2 µg/ml) and aminoglycosides (MIC50, > 8 µg/ml for gentamicin and MIC50, > 16 213

µg/ml for amikacin). All the A. baumannii isolates (n=14) resistant to carbapenems 214

carried the plasmid-borne carbapenemase gene blaOXA-24, or the insertion sequence 215

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ISAba1 upstream the chromosomally encoded blaOXA-51-like and/or AmpC (data not 216

shown). 217

The analysis of the nucleotide sequences of the seven PBP and one MTG genes in the A. 218

baumannii clinical isolates revealed specific hotspot mutation regions in all genes. 219

However, most of the allelic variations observed were silent mutations (data not 220

shown). Considering the sequences of the ATCC 17978 strain, the main changes in the 221

different PBP genes are shown in the Table 3. Although few mutations were found in 222

the amino acid sequences of the PBPs, none of these mutations could be related to β-223

lactams resistance. The observed changes in the amino acid sequence were more 224

associated to specific clonal patterns, and, in general, the same mutations were observed 225

in the distinct hospitals as well as in the already 10 sequenced genomes (Table 4). 226

Interestingly, the comparison of the amino acid consensus sequence of each PBP gene 227

of A. baumannii genomes deposited in the GenBank and amino acid consensus 228

sequence of the 26 A. baumannii clinical isolates showed that these genes were highly 229

conserved (identity of 99.6% to 100.0%). 230

In two isolates (HUMV-1319 and HUMV-5118) of an endemic carbapenems-resistant 231

clone (MIC > 32µg/ml) from one of the participating centers, PCR amplification of the 232

carboxypeptidase PBP6b showed a fragment of 2410 bp, instead of the expected 1320 233

bp. Sequencing of this amplicon indicated an IS disrupting the PBP6b gene. This IS was 234

identical to an IS30 family transposase found in the genome of the A. baumannii strain 235

ACICU and similar to ISAba125, only differing in a His222Tyr in the transposase gene. 236

Two imperfect inverted repeats (IR-L: aaacttgaagtcgaca and IR-R: tgtcgcacctcatgttt) 237

bordering the transposase gene were also identified. The IS was not found in the carO 238

gene in these two isolates (data not shown). 239

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DISCUSSION 240

PBPs are involved in the metabolism of peptidoglycan, an essential component of the 241

bacterial cell wall (33). β-lactams mimic the D-ala-D-ala dipeptide and act as suicide 242

inhibitors binding covalently to the PBPs (45). The inhibition of PBPs causes instability 243

in the cell wall, resulting in growth inhibition or lysis (42). In this study, seven PBP and 244

one MTG genes were found in the genomes of several A. baumannii strains: PBP1a, 245

PBP1b, PBP2, PBP3, PBP5/6, PBP6b, PBP7/8 and MtgA. The amino acid sequences of 246

these PBPs in the A. baumannii clinical isolates were compared with those present in 247

the strain ATCC 17978. Such strain was isolated in the early 1950s (1), prior to the 248

development of the majority of the antimicrobial agents used in the clinical practice. 249

This comparison showed that PBP genes are highly conserved in all A. baumannii 250

strains analyzed. The few point mutations observed could not be associated to 251

carbapenem resistance, as they were found in both susceptible and resistant strains. 252

Some point mutations were observed in specific isolates, especially in the genome of 253

SDF strain (Table 5), a multi-susceptible A. baumannii strain. It is most probably that 254

these variations are associated with clonal patterns. 255

PBPs with low affinity for β-lactams had been described in Acinetobacter spp. 256

(12,26,38). Gehrlein et al. (12) described in an A. baumannii clinical strain, seven PBPs 257

with the apparent molecular weights of 94, 84, 65, 61, 48, 40 and 24 kDa, based on 258

phenotypic assays, that could be assigned to PBP1a (94.74 kDa), 1b (88.26 kDa), 2 259

(74.42 kDa), 3 (67.66 KDa), 5/6 (48.84 KDa) and 6 (41.78 kDa), respectively, but the 260

last one do not correlate with PBP7/8 (36.86 KDa). They also observed that imipenem 261

could select in vitro for a resistant A. baumannii mutant showing a complex 262

reorganization of PBPs. As no alterations in outer membrane proteins or in β-lactamases 263

production were detected in the imipenem-resistant mutant, the authors associated the 264

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alterations in PBP profiles with the observed imipenem resistance. In another study, 265

Obara and Nakae (26) evaluated 12 imipenem-susceptible strains of A. calcoaceticus 266

and detected six PBP bands of 94 (PBPla), 92 (PBPlb), 86 (PBPlc), 74 (PBP2), 59 267

(PBP3) and 42 kDa (PBP4). In vitro selected mutants resistant to cefoxitin, 268

cefoperazone or ceftazidime presented reduced expression of porins, and alterations in 269

PBP expression and/or affinity for β-lactams. In another study, a nosocomial imipenem-270

resistant strain of A. baumannii was reported showing PBPs with low affinity for β-271

lactamases inhibitors, especially clavulanic acid (38). This study also showed that 272

sulbactam bound to PBPs better than tazobactam, even in imipenem-resistant strains, 273

what may justify the satisfactory in vitro activity of sulbactam against some multidrug-274

resistant A. baumannii isolates (22,28). 275

Fernández-Cuenca et al. (11) used 12% SDS-PAGE gels marked with 125I-ampicillin to 276

evaluate the PBP profiles of two groups of A. baumannii with MIC of imipenem and 277

meropenem of 0.25 to 2 µg/mL and 4 to 32 µg/mL, respectively. The isolates HUS-31 278

and HUS-457 included in the present study are representative of both groups, 279

respectively. Isolates with MICs of carbapenems ≥4 µg/mL showed a reduced 280

expression of a 73.2 kDa PBP [named PBP2, which may correspond to the PBP2 281

identified in this study (74.42 kDa)], associated with the production of several beta-282

lactamases, including oxacillinases (which has been confirmed in the present study) 283

and, in some isolates, with the loss of a 22.5 kDa porin. We have not observed any 284

mutations in PBP2 (or any other PBP) of strain HUS-457, suggesting that regulatory 285

mechanisms could be involved in the reported decreased expression of the 73.2 kDa 286

protein in this isolate. 287

An IS has been found to disrupt the dacD gene (encoding for the PBP6b) in two isolates 288

of a carbapenem-resistant endemic clone. This IS is similar to ISAba125, which was 289

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previously described disrupting the carO gene (25), coding for a porin associated with 290

resistance to carbapenems in A. baumannii. Although E. coli mutants lacking one or all 291

of the LMM PBPs failed to substantially affect cell division or elongation (13), Moya el 292

al. (24) have reported that in P. aeruginosa the inactivation of the dacB gene (encoding 293

the nonessential PBP4, but absent in A. baumannii) is associated with a complex high 294

level β-lactam resistance, triggering the overproduction of ampC and the specific 295

activation of the CreBC two-component regulator, that also activates the expression of 296

β-lactamase in a PBP4-like mutant of Aeromonas (36). Zamorano et al. (44) also 297

showed that the dacB inactivation produced significantly higher MICs of 298

antipseudomonal penicillins and cephalosporins than ampD inactivation. We may 299

speculate that the importance of the inactivation of the PBP6 gene in carbapenem 300

resistance might be marginal. It was observed in only two of the 14 carbapenem-301

resistant isolates we studied (both of them belonging to the same clone), in which 302

resistance can be explained by the production of OXA-24. In fact, other OXA-24-303

producing isolates are also resistant to carbapenems (Table 1) even they do not have an 304

inactivated PBP6 gene. Unfortunately, attempts to silence the dacD gene in the 305

carbapenem-susceptible strain of A. baumannii ATCC 19606 (to verify the actual role 306

of PBP6b in β-lactam resistance) were unsuccessful until now. Additional studies on 307

this topic are warranted. 308

Recently, Yun et al. (43) evaluated the proteome regulation in an imipenem-resistant A. 309

baumannii strain under antibiotic stress conditions. They observed that the levels of 310

RND family transporters (AdeABC and AdeJIK), the PBP genes ponA (PBP1a), ftsI 311

(PBP3) and dacC (PBP5/6), and noticeable the AmpC β-lactamase were increased in 312

the presence of imipenem. In opposite, a repression of OMPs OmpA and OmpW were 313

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observed in the same conditions. These results suggest that such mechanisms contribute 314

together to imipenem resistance in A. baumannii. 315

In the presence of efficient alternatives, as the CHDLs production associated or not with 316

porin loss, the selective pressure of β-lactams against PBPs in A. baumannii may be 317

insufficient to select for structural changes in these proteins directly involved in β-318

lactams resistance. On the other hand, it is possible that other complex mechanisms 319

associated with the regulation of PBP genes as well as post-transcriptional events could 320

cause the β-lactam resistance in this pathogen. Our findings may represent a first step in 321

understanding the actual role of the PBPs in the complex resistance of A. baumannii to 322

β-lactams. 323

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ACKNOWLEDGMENTS 324

This work was partially supported by Instituto de Salud Carlos III, Spanish Network for 325

Research in Infectious Diseases (REIPI, RD06/0008) and FIS (PI080209). We are 326

grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 327

(CAPES), which gave a PDEE grant to R.C. (protocol 4149/08-4). L. Dijkshoorn is 328

thanked for providing A. baumannii strains RUH-134 and RUH-875. We acknowledge 329

the funding of MICINN (BFU2009-09200) to J.A.A. 330

331

TRANSPARENCY DECLARATIONS 332

We have no conflicts of interest to report. 333

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Table 1 - Genetic characterization of the 26 A. baumannii clinical isolates. 484

MIC (µµµµg/ml) of:b Strain

Genomic

Identification

Hospitala/City PFGE

clone IPM MEM FEP CAZ CRO CTX ATM AMK GEN MIN CIP TGC CST

Oxacilinases types

HUMV-823 A. baumannii HUMV/Santander A >32 >32 >32 >32 >32 >32 >16 >16 >8 8 >2 1 0.5 OXA-51-like+ISAba1

HUMV-1175 A. baumannii HUMV/Santander A1 >32 >32 >32 >32 >32 >32 >16 >16 >8 8 >2 1 0.5 OXA-51-like+ISAba1

HUMV-3743 A. baumannii HUMV/Santander A2 >32 >32 >32 >32 >32 >32 >16 >16 >8 8 >2 1 0.5 OXA-51-like+ISAba1

HUMV-1102 A. baumannii HUMV/Santander B 2 2 8 16 >32 >32 16 ≤8 ≤4 4 >2 1 1 OXA-51-like

HUMV-2790 A. baumannii HUMV/Santander B 1 1 8 >32 >32 >32 >16 ≤8 ≤4 4 >2 1 1 OXA-51-like

HUMV-1319 A. baumannii HUMV/Santander C >32 >32 >32 16 >32 >32 >16 >16 >8 2 >2 1 0.5 OXA-51-like, OXA-24

HUMV-5118 A. baumannii HUMV/Santander C >32 >32 >32 >32 >32 >32 >16 >16 >8 1 >2 1 0.5 OXA-51-like, OXA-24

HUMV-2471 A. baumannii HUMV/Santander D >32 >32 8 ≤8 4 4 8 >16 >8 2 >2 0.25 0.25 OXA-51-like, OXA-24

HUMV-4066 A. baumannii HUMV/Santander D >32 >32 >32 ≤8 32 32 >16 >16 >8 2 >2 0.25 0.5 OXA-51-like, OXA-24

HUMV-6457 A. baumannii HUMV/Santander D >32 >32 >32 16 8 8 16 ≤8 ≤4 2 >2 0.25 0.5 OXA-51-like, OXA-24

HUMV-2120 A. baumannii HUMV/Santander E 0.25 0.25 4 ≤8 8 8 16 ≤8 ≤4 1 ≤0.125 0.5 0.5 OXA-51-like

HUMV-4674 A. baumannii HUMV/Santander F 0.5 0.5 8 ≤8 8 8 16 ≤8 ≤4 0.5 1 2 0.5 OXA-51-like

HC-360 A. baumannii HC/Barcelona G 1 2 32 32 >32 >32 >16 >16 >8 2 >2 1 0.25 OXA-51-like

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HC-3581 A. baumannii HC/Barcelona G 1 2 32 32 >32 >32 >16 >16 >8 2 >2 1 0.25 OXA-51-like

HC-4249 A. baumannii HC/Barcelona G >32 >32 >32 32 >32 >32 >16 16 >8 <0.5 >2 0.5 0.5 OXA-51-like, OXA-24

HC-4275 A. baumannii HC/Barcelona G >32 >32 >32 32 >32 >32 >16 >16 >8 1 >2 1 0.125 OXA-51-like+ISAba1, OXA-24

HC-60 A. baumannii HC/Barcelona H 1 4 32 ≤8 16 16 >16 16 >8 <0.5 1 1 0.125 OXA-51-like

HC-3343 A. baumannii HC/Barcelona H 1 4 32 ≤8 16 16 >16 16 >8 <0.5 1 1 0.125 OXA-51-like

HC-4256 A. baumannii HC/Barcelona H >32 >32 16 ≤8 32 32 >16 ≤8 >8 <0.5 1 0.5 0.25 OXA-51-like, OXA-24

HC-3202 A. baumannii HC/Barcelona H >32 >32 32 32 >32 >32 >16 >16 >8 2 >2 1 0.25 OXA-51-like, OXA-24

HC-771 A. baumannii HC/Barcelona I 1 4 16 >32 >32 >32 >16 16 >8 8 >2 0.5 0.25 OXA-51-like

HC-769 A. baumannii HC/Barcelona I 1 8 16 >32 >32 >32 >16 >16 >8 8 >2 1 0.25 OXA-51-like

HC-181 A. baumannii HC/Barcelona I >32 >32 16 >32 >32 >32 >16 >16 >8 8 >2 0.5 0.25 OXA-51-like+ISAba1

HC-1959 A. baumannii HC/Barcelona I >32 >32 >32 >32 >32 >32 >16 >16 8 8 >2 0.5 0.25 OXA-51-like+ISAba1

HUS-31 A. baumannii HUVM/Seville J 0.25 0.5 32 16 >32 >32 >16 >16 >8 2 >2 2 0.25 OXA-51-like

HUS-457 A. baumannii HUVM/Seville K 4 4 4 16 32 32 >16 16 >8 <0.5 >2 0.5 0.125 OXA-51-like

a. HUMV - Hospital Universitario Marqués de Valdecilla, HC - Hospital Clinic, HUVM - Hospital Universitario Virgen Macarena. 485

b. IPM, imipenem; MEM, meropenem; FEP, cefepime; CAZ, ceftazidime; CRO, ceftriaxone; CTX, cefotaxime; ATM, aztreonam; AMK, 486

amikacin; GEN, gentamicin; MIN, minocycline; CIP, ciprofloxacin; TGC, tigecycline; CST, colistin. 487

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Table 2 - The penicillin-binding proteins (PBPs) of A. baumannii. 488

% Identity

PBP (gene) PBPs classificationa Molecular function Possible physiological

functionb Pseudomonas

aeruginosa

Escherichia

coli

PBP1a (ponA) HMM Class A (subclass A1) Transglycosylase and

transpeptidase Peptidoglycan synthesis 43 36

PBP1b (mrcB) HMM Class A (subclass A2) Transglycosylase and

transpeptidase Peptidoglycan synthesis 42 32

MtgA (mtgA) Monofunctional enzymes

(MGTs) Monofunctional transglycosylase

Unknown 34 32

PBP2 (mrdA/pbpA) HMM Class B (Subclass B2) Transpeptidase Cell elongation 45 39

PBP3 (ftsI) HMM Class B (Subclass B3) Transpeptidase Septum formation

(Cell division) 39 39

PBP5/6 (dacC) LMM Class C (Type-5) D-ala-D-ala-carboxypeptidase Unknown 48 40

PBP6b (dacD) LMM Class C (Type-5) D-ala-D-ala-carboxypeptidase Unknown - 31

PBP7/8 (pbpG) LMM Class C (Type-7) Endopeptidase Unknown 38 34

a. HMM, High molecular mass; LMM, Low molecular mass. 489

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b. Enzymatic activities and functions predicted by sequence homology analysis. 490

c. Amino acid sequence homology for the PBP and MGT proteins from the genomes of the P. aeruginosa PA7 strain (accession no. 491

NC_009656), E. coli O157:H7 strain EDL933 (accession no. NC_002655.2) and A. baumannii SDF strain (accession no. NC_010400). 492

Analysis was performed using the CLUSTAL W2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). 493

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Table 3 - Point mutations observed in the PBP genes in susceptible and resistant A. baumannii strains to carbapenems. 494

Point Mutationsc,d

PBP1a PBP1b PBP2 PBP3 PBP5/6 PBP6b PBP7/8 MtgA Strainsa,b

ponA mrdA pbpA fstl dacC dacD pbpG mtgA

HUMV (Imipenem-susceptible)

L147I (B), T636A (E)

P112S (B) P665A (E) 0 e

N329S (B), T374V (B, E), N296D (B, E), N307S (B, E)

P28S (B), T188P (B)

T45S (B, F), A84T (B)

F18L (B, E, F) , T49P (B, E, F),

I54V (E, F), N179S (B, E, F)

HUMV (Imipenem-resistant)

A244T (C), S382N (C),

T636A (C, D)

P112S (A), P764S (C)

V509I (C), E110Q (D)

G523V (C)

N329S (A, C, D), T374V (A), N296D (A), N307S (A)

P28S (A), A277T (D), V350I (D), S429N (D)

T45S (A, C, D), A84T (A)

F18L (A, C, D), T49P (A, C, D),

Q100E (C), N179S (A, C, D)

HC (Imipenem-susceptible)

T38A (H), L147I (I),

A244T (G), S382N (G), T636A (G)

P112S (I), P764S (G)

V509I (G) G523V (G), H370Y (I)

N329S (I) P28S (I),

T188P (G)

T39I (I), T45S (G, H, I),

A84T (I)

F18L (G, H, I), T49P (G, I), Q100E (G),

N179S (G, H, I)

HC (Imipenem-resistant)

L147I (I), A244T (G, H), S382N (G, H), T636A (G, H)

P112S (I), P764S (G, H)

V509I (G) G523V (G), H370Y (I)

N329S (I) P28S (I),

T188P (G, H)

T39I (I), T45S (G, H, I),

A84T (I)

F18L (G, H, I), T49P (G, I),

Q100E (G, H), N179S (G, H, I)

HUVM (Imipenem-susceptible)

A244T (J), S382N (J), T636A (J)

P764S (J) V509I (J), E110Q (K)

G523V (J) 0 A277T (K), V350I (K), S429N (K)

T45S (J, K)

F18L (J, K), T49P (J, K), Q100E (J),

N179S (J, K)

a. HUMV - Hospital Universitario Marqués de Valdecilla, HC - Hospital Clinic, HUVM - Hospital Universitario Virgen Macarena. 495

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b. Imipenem-susceptible (MIC ≤ 4 µg/mL) and Imipenem-resistant (MIC ≥ 16 µg/mL). 496

c. Amino acid substitutions are labelled with respect to A. baumannii strain ATCC 17978. 497

d. Letters in brackets represents clonal pattern. 498

e. No mutations have been observed when compared with the genome of A. baumannii strain ATCC 17978. 499

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Table 4 - Point mutations observed in the PBP genes of the 10 A. baumannii genomes and the reference strain RUH-134. 500

Point Mutations

PBP1a PBP1b PBP2 PBP3 PBP5/6 PBP6b PBP7/8 MtgA Strain

ponA mrdA pbpA fstl dacC dacD pbpG mtgA

RUH-134 L147I 0a 0 0 N329S P28S A84T F18L, T49P

ACICU L147I P112S 0 A346V, H370Y 0 P28S, K229Q T45S, A84T F18L, T49P,

N179S

SDF A224T, T636A S274A, R590H, Q601E, R712H,

Q765E 0 0 S5N S418N T45S, R218H

V8M, F18M, T49P, Q100E,

N179S

AYE T38A, A244T N513H P665A 0 0 0 T45S F18L, T49P,

Q100E, N179S

AB0057 T38A, A244T N513H P665A 0 0 0 T45S F18L, T49P,

Q100E, N179S

AB307-0294 T38A, A244T,

A613T N513H P665A 0 N329S 0 T45S

F18L, T49P, Q100E, N179S

AB900 T636A 0 E110Q 0 0 A277T, V350I,

S429N T45S

F18L, T49P, N179S

AB056 T38A, A244T,

T776A 0 P665A 0 0 0 T45S

F18L, T49P, Q100E, N179S

AB058 T38A, A244T,

T776A 0 P665A V565L 0 0 T45S

F18L, T49P, Q100E, N179S

AB059 T38A, A244T,

T776A 0 P665A 0 0 0 T45S

F18L, T49P, Q100E, N179S

ATCC 19606 V623I 0 0 0 0 Q143K T45S F18L, T49P,

N179S

a. No mutations have been observed when compared with the A. baumannii ATCC 17978 genome. 501

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FIGURE LEGENDS 502

FIG 1. Identification of the conserved motifs in the consensus sequences of the HMM 503

PBP genes of A. baumannii genomes used for the PBPs classification, according to 504

Sauvage et al. (33). A - The localization of the five characteristic conserved motifs of 505

the N-terminal domain in both HMM class A PBP1a and 1b are showed by their 506

respective boxes; B - Differences in the motif 3 of the C-terminal domain, between the 507

HMM of Class A and class B PBPs are showed by the black arrows; C - A residue of 508

motif 2 in third position in the active site of the HMM of class B PBPs used to divide 509

these PBPs in subclasses B2 and B3 are signalized by the black arrow. 510

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FIG 1. 511

A

B

Motif 1

Motif 2 Motif 3

Motif 4

Motif 5

C

512

513

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A

B

Motif 1

Motif 2 Motif 3

Motif 4

Motif 5

C

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