Characterisation of fosfomycin resistance mechanisms and molecular epidemiology in extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates

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Q2 Short Communication

Characterisation of fosfomycin resistance mechanisms and molecular epidemiology in extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates Q1 Po-Liang Lu a,b,c, Ya-Ju Hsieh d, Jun-En Lin e, Jun-Wei Huang e, Tsung-Ying Yang e, Lin Lin f,

Sung-Pin Tseng e,g,* a

Department of Laboratory Medicine, Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan c Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan d Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan f Department of Culinary Art, I-Shou University, Kaohsiung, Taiwan g Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan b

A R T I C L E

I N F O

Article history: Received 25 March 2016 Accepted 8 August 2016 Keywords: Fosfomycin resistance mechanism murA glpT uhpT Transporters

A B S T R A C T

Although fosfomycin is a treatment option for infections caused by extended-spectrum β-lactamase (ESBL)producing Enterobacteriaceae, fosfomycin resistance has been documented. To our knowledge, fosfomycin resistance mechanisms in Klebsiella pneumoniae have not been systematically investigated. A total of 108 ESBL-producing K. pneumoniae isolates collected from Kaohsiung Medical University Hospital, Taiwan, from August 2012 to May 2013 were analysed in this study. Pulsed-field gel electrophoresis (PFGE) revealed 64 pulsotypes and six non-typeable isolates, indicating high genetic diversity. Moreover, pulsotypes V (n = 6), VII (n = 11) and LI (n = 4) belonging to ST11 were major types. Among 30 (27.8%) fosfomycin-nonsusceptible isolates, 21 (70%) had a MurA amino acid substitution, and seven new variations increased the fosfomycin minimum inhibitory concentration (MIC) by 8- to 16-fold compared with wild-type MurA in Escherichia coli DH5α.strain. Functionless transporters (GlpT and UhpT) with various mutations were found in 29 isolates (97%). No known fosfomycin-modifying enzymes were detected in this study. The major resistance mechanisms to fosfomycin in K. pneumoniae were amino acid variations in the drug target and transporters. © 2016 Published by Elsevier B.V.

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1. Introduction Emerging problems caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been reported worldwide [1,2]. Infections caused by ESBL-producing Enterobacteriaceae are difficult to treat and have become a major clinical problem. Fosfomycin is a viable treatment option for patients with ESBLproducing Enterobacteriaceae infections [3,4]. In 2010, Falagas et al reported the satisfactory susceptibility of ESBL-producing Escherichia coli (96.8%; 1604/1657) and ESBL-producing Klebsiella pneumoniae (81.3%; 608/748) to fosfomycin [3]. These reports showed that the old antibiotic fosfomycin had the capacity to treat ESBL-producing Enterobacteriaceae infections. Fosfomycin resistance mechanisms include modification of the antibiotic target (MurA), transporter inactivation (GlpT and UhpT

2. Materials and methods

59 60 61 62 63 64

transporters and their regulating genes, such as uhpA, cyaA and ptsI) and antibiotic inactivation (FosA and FosC) in E. coli [5]. However, only a few studies have described fosfomycin resistance mechanisms that include fosfomycin-inactivating enzymes [glutathione-S-transferase (GST), fosA3 and fosK96] [6–8] and reduction in the permeability of the cell membrane [8] in K. pneumoniae. The relationship between fosfomycin resistance and variations in the target gene (murA) or functionless transporters (GlpT and UhpT) has not been reported for K. pneumoniae. Besides, only one study has indicated that 28 (42.4%) of 66 ESBL-producing K. pneumoniae isolates exhibited fosfomycin resistance, whereas the resistance mechanism remains unknown in Taiwan [9]. In the present study, we aimed to investigate the activity of fosfomycin, its resistance mechanisms and its molecular epidemiology in ESBL-producing K. pneumoniae isolates in Taiwan.

* Corresponding author. Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung, Taiwan. Fax: +886 7 311 3449. E-mail address: [email protected] (S.-P. Tseng).

2.1. Bacterial isolates During the period from August 2012 to May 2013, a total of 108 ESBL-producing K. pneumoniae isolates were collected from

http://dx.doi.org/10.1016/j.ijantimicag.2016.08.013 0924-8579/© 2016 Published by Elsevier B.V.

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86 Kaohsiung Medical University Hospital in Taiwan. The sources of 87 the isolates were urine (n = 47), sputum (n = 26), abscess (n = 8), blood 88 (n = 5), pus (n = 3) and other patient samples (n = 19). 89 90 2.2. Antimicrobial susceptibility testing 91 92 Antimicrobial susceptibility testing was performed using the agar 93 dilution method according to the guidelines of the Clinical and Lab94 oratory Standards Institute (CLSI) [10]. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of an95 tibiotic that prevented bacterial growth after 16–20 h of incubation 96 at 37 °C. The following antibiotics were tested: ampicillin; amikacin; 97 tetracycline; levofloxacin; chloramphenicol; fosfomycin (supple98 mented with 25 μg/mL glucose-6-phosphate); cefotaxime; and 99 meropenem. 100 101 2.3. Genotyping by pulsed-field gel electrophoresis (PFGE) and 102 multilocus sequence typing (MLST) 103 104 Epidemiological investigation was performed by PFGE and MLST. 105 Pulsotypes were determined according to previously described 106 methods [11]. The MLST scheme of K. pneumoniae uses internal frag107 ments from seven housekeeping genes, and primers were derived 108 109 Q4 from the K. pneumoniae MLST database (http://bigsdb.web.pasteur.fr/ klebsiella/). PCR amplification and sequencing were performed fol110 lowing protocols suggested on this website. Three isolates (12-J3, 111 11-G6 and K68) belonging to pulsotypes V, VII and LI, respective112 ly, were analysed by MLST. 113 114 2.4. Detection of antimicrobial resistance genes 115 116 Plasmid DNA was extracted using a QIAGEN Plasmid Mini 117 Kit (QIAGEN, Valencia, CA). For the detection of ESBLs (blaSHV, 118 blaCTX-M-group 1, blaCTX-M-group 2 and blaCTX-M-group 9), the plasmid-mediated 119 AmpC genes blaDHA and blaCMY, primer sets from previous reports 120 were used [6,11]. The primer sets listed in Supplementary Table S1 121 were used to detect fosfomycin resistance genes (fosA, fosA3, fosC2 122 and fosKP96), the fosfomycin target (MurA), and functionless trans123 porters (GlpT and UhpT) and their regulating genes (uhpA and ptsI). 124 Each plasmid sample was verified by PCR of the rpoB gene (a chro125 mosomal gene); if the PCR result was positive, the plasmid sample 126 was contaminated by chromosomal DNA and was discarded. 127 128 2.5. Use of carbohydrates 129 130 The use of carbohydrates was analysed as in our previous report 131 [11]. Briefly, 0.2% (w/v) glycerol 6-phosphate (G6P) or sn-glycerol 132 3-phosphate (G3P) was supplied as the sole carbon source in M9 133 minimal medium agar. An overnight bacterial suspension was 134 washed with an equivalent volume of saline and the turbidity was 135 adjusted to match a McFarland no. 4 standard. Bacterial suspen136 sions in 200 μL were plated on M9 minimal medium agar 137 supplemented with different sole carbohydrates at 37 °C for 48 h. 138 Negative growth was identified by the absence of colonies on the 139 agar surface. 140 141 2.6. Sequence analysis of fosfomycin-related chromosomal genes 142 143 PCR and sequencing were performed to amplify the entire se144 quences of murA, glpT, uhpT, uhpA and ptsI using the primers listed 145 in Supplementary Table S1. Variations in amino acids were com146 pared with the fosfomycin-susceptible strain K. pneumoniae K68. 147 The nucleotide sequences of murA, glpT, uhpT, uhpA and ptsI from 148 K. pneumoniae K68 were deposited in GenBank under accession 149 nos. KT334183, KT334186, KT334184, KT334185 and KT334187, 150 respectively. 151

2.7. Cloning overexpressed MurA with different amino acid variations The murA gene (K68, 12-A5, 12-I3, 12-I4, 13-A2, 13-A7, K121 and K154) was PCR-amplified using a cloning primer (Supplementary Table S1). PCR products were cloned into a T&A™ plasmid (Yeastern Biotech, Taipei, Taiwan) and were transformed into E. coli DH5α strain. Insertion of murA was verified by determining the nucleotide sequence. To determine the fosfomycin susceptibility of murAoverexpressing E. coli strains, 25 μg/mL G6P and 1 mM isopropyl β-d1-thiogalactopyranoside (IPTG) were added to Muller–Hinton agar with various concentrations of fosfomycin (0–1024 μg/mL). 3. Results 3.1. Antimicrobial susceptibility testing and molecular typing The results of antimicrobial susceptibility testing are presented in Supplementary Table S2. All 108 ESBL-producing isolates were resistant to ampicillin and cefotaxime. The resistance rate for tetracycline was 89.8%, and the rates for amikacin, levofloxacin and chloramphenicol were 62.0%, 64.8% and 56.5%, respectively. The susceptibility rates for fosfomycin and meropenem were 72.2% (78/ 108) and 97.2% (105/108), respectively. PFGE analysis revealed 64 pulsotypes and six non-typeable isolates, indicating the genetic diversity of ESBL-producing isolates (Fig. 1). Pulsotypes V (n = 6), VII (n = 11) and LI (n = 4) belonging to ST11 were the predominant pulsotypes, indicating clonal spread. Among these three pulsotypes, the percentage of fosfomycin-non-susceptible K. pneumoniae isolates was 17% (1/6) in pulsotype V, 36% (4/11) in pulsotype VII and 25% (1/4) in pulsotype LI. The distribution of ESBL types is shown in Supplementary Table S3; however, no specific pulsotype was related to resistance to fosfomycin. 3.2. Resistance mechanisms of fosfomycin-non-susceptible Klebsiella pneumoniae isolates Among 108 ESBL-producing K. pneumoniae isolates, 30 (27.8%) were not susceptible to fosfomycin (Table 1). However, fosfomycinmodifying enzymes, including fosA, fosA3, fosC and fosK96, were not discovered in any of the fosfomycin-non-susceptible K. pneumoniae isolates. Of the 30 fosfomycin-non-susceptible isolates, 21 (70%) had amino acid substitutions in MurA (Table 1). The seven variations in MurA included Gly118Asp (n = 3), Thr214Ile (n = 3), Thr287Asn (n = 9) and Thr307Lys (n = 3) as well as three double mutants (Glu130Lys Leu282Phe; Asp259Asn Arg267Leu; Asp260Tyr Thr287Asn; n = 1 for each). To test the correlation between these amino acid substitutions and fosfomycin resistance, wild-type and mutant MurA were transconjugated for expression into E. coli DH5α (Table 2). Variations in MurA increased the MICs by 8- to 16-fold compared with wild-type MurA. These results indicated that amino acid substitutions in MurA were associated with fosfomycin resistance. A previous study reported that defective UhpT/GlpT transporters leading to fosfomycin-resistant isolates did not grow in minimal medium agar supplemented with G6P/G3P, respectively [5]. In the current study, nine isolates in which MurA was not mutated did not grow on minimal medium agar supplemented with G3P and seven isolates did not grow on minimal medium agar supplemented with G6P (Table 1). These results indicated that the UhpT and/or GlpT transporter could be defective in these isolates. To determine the resistance mechanism, the nucleotide sequences of glpT, uhpT, uhpA and ptsI were tested for all of the non-susceptible isolates. Among the nine isolates that did not grow on agar supplemented with G3P, six isolates (K163, K63, K47, K131, 13-A8 and K136) had various amino acid substitutions in GlpT (Table 1). K63 and K136 had single

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Fig. 1. Dendrogram of pulsotype relationships developed through the unweighted pair-group method with arithmetic mean (UPGMA) using BioNumerics v.6.5 (Applied Maths, Sint-Martens-Latem, Belgium). Pulsotypes were assigned to the same clusters if they exhibited 80% similarity in the dendrogram. Information of multilocus sequence typing (MLST), susceptibility to fosfomycin and extended-spectrum β-lactamases ESBLs were determined in three clonal clusters (V, VII and LI). S, susceptible; I, intermediate; R, resistant.

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amino acid substitutions at codons 266 (Ile266Ser) and 278 (Asn278Lys), respectively. Double substitutions were found in four isolates: K163 (Pro305Ala and Arg317His); K47 (Ile266Ser and Asn278Lys); K131 (Pro257Arg and Pro327Thr); and 13-A8 (Ala318Thr and Leu338Trp). Among the seven isolates that did not grow on agar supplemented with G6P, a single amino acid substitution in UhpT was found in K155 (Arg323Lys) and K37 (Gln320Lys). The K166 isolate did not have any amino acid substitutions in the glpT and uhpT genes, but variations were found in the upstream regulator ptsI (Asn30Asp, Arg38Gly and Tyr57Glu). 4. Discussion ST11 was the epidemic clone associated with ESBLs in Europe and Asia countries [12,13]. Three major pulsotypes (V, VII and LI) belonging to ST11 (Fig. 1) indicated that ST11 was the predominant type of ESBL-producing K. pneumoniae in Taiwan.

Most studies have revealed favourable susceptibility of ESBLproducing K. pneumoniae isolates to fosfomycin, such as those conducted in Thailand (90.7%; 39/43) [14] and Spain (97%; 134/ 138) [15], and multidrug-resistant K. pneumoniae containing ESBL and metallo-β-lactamase in Greece (100%; 30/30) [16]. However, recent studies have demonstrated that the susceptibility rate to fosfomycin is lower in ESBL-producing K. pneumoniae isolates [9,17]. Demir et al [17] and Liu et al [9] reported that 66.1% and 57.6% of ESBL-producing K. pneumoniae isolates were resistant to fosfomycin in Turkey and northern Taiwan, respectively. The current study revealed that 72.2% (78/108) of ESBL-producing K. pneumoniae isolates were susceptible to fosfomycin in southern Taiwan (Supplementary Table S2). These data indicate that the non-susceptibility rate to fosfomycin in ESBL-producing K. pneumoniae isolates is higher in Taiwan than in other countries. Only a few studies have described fosfomycin-inactivating enzymes (GST, fosA3 and fosK96) in K. pneumoniae [6–8]. Furthermore,

Please cite this article in press as: Po-Liang Lu, et al., Characterisation of fosfomycin resistance mechanisms and molecular epidemiology in extended-spectrum β-lactamaseproducing Klebsiella pneumoniae isolates, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.08.013

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Table 1 Characteristics of 30 fosfomycin-non-susceptible Klebsiella pneumoniae isolates and the fosfomycin-susceptible control strain K. pneumoniae K68. Strain

261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

MIC (μg/mL) +G6P

260

Growth −G6P

Amino acid substitutions

G3P

G6P

MurA

GlpT

UhpT

K68 (control) 16 32 Intermediate susceptibility to fosfomycin (n = 19) 12-A5 128 256

+

+

None

None

None

+

−

Gly118Asp

None

12-G10 12-J3

128 128

256 256

− −

+ +

Gly118Asp Gly118Asp

12-G4 13-A7

128 128

256 256

+ −

− +

Thr214Ile Thr214Ile

12-B8

128

256

−

−

Thr287Asn

12-B10 12-F1 12-F4

128 128 128

256 256 256

− + −

+ − +

Thr287Asn Thr287Asn Thr287Asn

12-G1

128

256

−

+

Thr287Asn

12-H8

128

256

−

−

Thr287Asn

12-I3

128

256

−

+

Thr287Asn

13-A1 12-I4

128 128

256 256

+ −

− +

13-A2

128

256

−

+

K163

128

256

−

+

Thr287Asn Glu130Lys Leu282Phe Asp259Asn Arg267Leu None

K63 K47

128 128

256 256

− −

+ −

None None

K131

128

256

−

−

None

Ala318Thr Arg177Lys Leu338Trp None Phe236Pro Arg344Gly Leu338Trp Leu338Trp Phe183Leu Cys278Gly None Ala318Thr Leu338Trp Ala255Glu Leu338Trp Asp214Glu Ala318Thr Leu338Trp Ala255Glu None Ile226Thr Arg177Lys Phe184bpIle Ile226Thr Pro305Ala Arg317His Ile266Ser Ile266Ser Asn278Lys Pro257Arg Pro327Thr

Arg171Vla Arg312Pro None None

Resistant to fosfomycin (n = 11) 12-J1 256

512

+

−

Thr214Ile

None

12-J7

256

512

−

+

Thr287Asn

K11 K144

256 256

512 512

− −

+ +

Thr307Lys Thr307Lys

K121 K154

512 512

1024 1024

− −

− +

Thr307Lys Asp260Tyr Thr287Asn

13-A8

256

512

−

−

None

K136 K166 K155 K37

256 256 512 512

512 512 1024 1024

− − − −

− − − −

None None None None

Cys221Arg Glu241Lys Asn278Lys Ser205Thr Arg206Lys Thr208Ser Asn278Lys Asn278Lys Ile266Ser Ser283Cys Ile293Phe Gly300Arg Ala318Thr Leu338Trp Asn278Lys None None None

Ala301Gly None

Arg312Pro None Arg171Vla None None Lys286Arg Ala301Gly None Gly196Glu None None Ala252Pro None None Ala301Gly Glu317Lys Arg171Vla Leu178Phe None None None

None Ser266Pro Ile283Leu

Arg165Gly Arg312Pro Ala301Gly None Arg323Lys Gln320Lys

MIC, minimum inhibitory concentration; G6P, glucose 6-phosphate; G3P, sn-glycerol 3-phosphate.

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the correlation between fosfomycin resistance and murA or functionless transporters (GlpT and UhpT) is not reported in the literature. In this study, 21 (70%) of 30 fosfomycin-non-susceptible isolates demonstrated an amino acid substitution in MurA (Table 1). Susceptibility to fosfomycin was 8- to 16-fold higher in isolates with seven new variations in MurA compared with isolates with wildtype MurA. It is possible that this effect is mediated by the binding of fosfomycin to the Cys115 residue (active site) or three conserved positively charged residues (Lys22, Arg120 and Arg397) in MurA [18]. In addition, functionless transporters (GlpT and UhpT) or its regulating gene with various mutations were found in 29

isolates (97%) (Table 1). These amino acid substitutions could be associated with fosfomycin resistance in K. pneumoniae. Nilsson et al reported that amino acid substitutions in transporter or regulating genes reduce the uptake of fosfomycin leading to fosfomycin resistance in E. coli isolates [19]. Mutations in cyaA (five isolates) and ptsI (two isolates) genes impaired both glpT and uhpT expression. In addition, mutations were present in uhpT and glpT genes in three and one isolates, respectively. Takahata et al also demonstrated different amino acid substitutions in GlpT and UhpT transporters in E. coli isolates [20]. Mutation in the glpT gene (four isolates) and the loss of the entire uhpT gene (two isolates) reduced

Please cite this article in press as: Po-Liang Lu, et al., Characterisation of fosfomycin resistance mechanisms and molecular epidemiology in extended-spectrum β-lactamaseproducing Klebsiella pneumoniae isolates, International Journal of Antimicrobial Agents (2016), doi: 10.1016/j.ijantimicag.2016.08.013

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Table 2 Fosfomycin minimum inhibitory concentrations (MICs) in Escherichia coli DH5α cells overexpressing wild-type and mutant murA genes.

344

Strain

Enzyme overexpressed

MIC (μg/mL)a

345 346 347 348 349 350 351 352 353 354 355 356

DH5α DH5α/murA (K68 control) DH5α/murA (12-A5) DH5α/murA (12-I3) DH5α/murA (12-I4)

None MurA (wild-type) MurA (Gly118Asp) MurA (Thr287Asn) MurA (Glu130Lys, Leu282Phe) MurA (Asp259Asn,Arg267Leu) MurA (Thr214Ile) MurA (Thr307Asn) MurA (Asp260Tyr, Thr287Asn)

0.25 16 128 128 128

357 358

DH5α/murA (13-A2) DH5α/murA (13-A7) DH5α/murA (K121) DH5α/murA (K154)

128 256 128 256

a MICs were determined in the presence of 1 mM isopropyl β-d-1thiogalactopyranoside (IPTG) to induce MurA expression.

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fosfomycin uptake into bacterial cells, thereby inducing fosfomycin resistance. The results of the current study revealed similar mechanisms in fosfomycin-resistant K. pneumoniae. 5. Conclusions In this study, 72.2% (78/108) of ESBL-producing K. pneumoniae isolates were susceptible to fosfomycin. The major resistance mechanisms to fosfomycin were amino acid variations in the GlpT and UhpT transporters and regulatory genes (uhpA and ptsI) (29/30; 97%) as well as modification of the target gene (murA) (21/30; 70%). Funding: This work was supported by the Ministry of Science and Technology [MOST 103-2320-B-037-023 and 105-2320-B-037003], Kaohsiung Medical University Research Foundation [KMUQ5 M105014] and Kaohsiung Medical University Hospital, Taiwan. Competing interests: None declared. Ethical approval: The Institutional Review Board of Kaohsiung Medical University Chung-Ho Memorial Hospital (Kaohsiung, Taiwan) waived the need for informed consent and approved this study [KMUHIRB-E-20150040]. Appendix. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2016.08.013. References [1] Pitout JD, Laupland KB. Extended-spectrum β-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008;8:159–66.

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[2] Zahar JR, Lortholary O, Martin C, Potel G, Plesiat P, Nordmann P. Addressing the challenge of extended-spectrum β-lactamases. Curr Opin Investig Drugs 2009;10:172–80. [3] Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum β-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis 2010;10:43–50. [4] Raz R. Fosfomycin: an old–new antibiotic. Clin Microbiol Infect 2012;18:4–7. [5] Castañeda-García A, Blázquez J, Rodríguez-Rojas A. Molecular mechanisms and clinical impact of acquired and intrinsic fosfomycin resistance. Antibiotics (Basel) 2013;2:217–36. [6] Ho PL, Chan J, Lo WU, Lai EL, Cheung YY, Lau TC, et al. Prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes among blood and urinary Escherichia coli isolates. J Med Microbiol 2013;62:1707–13. [7] Lee SY, Park YJ, Yu JK, Jung S, Kim Y, Jeong SH, et al. Prevalence of acquired fosfomycin resistance among extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26composite transposon surrounding fosA3. J Antimicrob Chemother 2012; 67:2843–7. [8] O’Hara K. Two different types of fosfomycin resistance in clinical isolates of Klebsiella pneumoniae. FEMS Microbiol Lett 1993;114:9–16. [9] Liu HY, Lin HC, Lin YC, Yu SH, Wu WH, Lee YJ. Antimicrobial susceptibilities of urinary extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae to fosfomycin and nitrofurantoin in a teaching hospital in Taiwan. J Microbiol Immunol Infect 2011;44:364–8. [10] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-fifth informational supplement. Wayne (PA): CLSI; 2015. Document M100-S25. [11] Tseng SP, Wang SF, Kuo CY, Huang JW, Hung WC, Ke GM, et al. Characterization of fosfomycin resistant extended-spectrum β-lactamase-producing Escherichia coli isolates from human and pig in Taiwan. PLoS ONE 2015;10:e0135864. [12] Damjanova I, Toth A, Paszti J, Hajbel-Vekony G, Jakab M, Berta J, et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type β-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? J Antimicrob Chemother 2008;62:978– 85. [13] Lee MY, Ko KS, Kang CI, Chung DR, Peck KR, Song JH. High prevalence of CTX-M-15-producing Klebsiella pneumoniae isolates in Asian countries: diverse clones and clonal dissemination. Int J Antimicrob Agents 2011;38:160–3. [14] Tharavichitkul P, Khantawa B, Bousoung V, Boonchoo M. Activity of fosfomycin against extended-spectrum-β-lactamase-producing Klebsiella pneumoniae and Escherichia coli in Maharaj Nakorn Chiang Mai Hospital. J Infect Dis Antimicrob Agents 2005;22:121–6. [15] de Cueto M, Lopez L, Hernandez JR, Morillo C, Pascual A. In vitro activity of fosfomycin against extended-spectrum-β-lactamase-producing Escherichia coli and Klebsiella pneumoniae: comparison of susceptibility testing procedures. Antimicrob Agents Chemother 2006;50:368–70. [16] Falagas ME, Kanellopoulou MD, Karageorgopoulos DE, Dimopoulos G, Rafailidis PI, Skarmoutsou ND, et al. Antimicrobial susceptibility of multidrug-resistant Gram negative bacteria to fosfomycin. Eur J Clin Microbiol Infect Dis 2008; 27:439–43. [17] Demir T, Buyukguclu T. Evaluation of the in vitro activity of fosfomycin tromethamine against Gram-negative bacterial strains recovered from community- and hospital-acquired urinary tract infections in Turkey. Int J Infect Dis 2013;17:e966–70. [18] Skarzynski T, Mistry A, Wonacott A, Hutchinson SE, Kelly VA, Duncan K. Structure of UDP-N-acetylglucosamine enolpyruvyl transferase, an enzyme essential for the synthesis of bacterial peptidoglycan, complexed with substrate UDP-N-acetylglucosamine and the drug fosfomycin. Structure 1996;4:1465–74. [19] Nilsson AI, Berg OG, Aspevall O, Kahlmeter G, Andersson DI. Biological costs and mechanisms of fosfomycin resistance in Escherichia coli. Antimicrob Agents Chemother 2003;47:2850–8. [20] Takahata S, Ida T, Hiraishi T, Sakakibara S, Maebashi K, Terada S, et al. Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli. Int J Antimicrob Agents 2010;35:333–7.

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