Dissemination of the Fosfomycin Resistance Gene fosA3 with CTX-M �-Lactamase Genes and rmtB Carried on IncFII Plasmids among Escherichia coli Isolates from Pets in China Jianxia Hou, Xianhui Huang, Yuting Deng, Liangying He, Tong Yang, Zhenling Zeng, Zhangliu Chen, and Jian-Hua Liu College of Veterinary Medicine, National Reference Laboratory of Veterinary Drug Residues, South China Agricultural University, Guangzhou, China The presence and characterization of plasmid-mediated fosfomycin resistance determinants among Escherichia coli isolates col- lected from pets in China between 2006 and 2010 were investigated. Twenty-nine isolates (9.0%) were positive for fosA3, and all of them were CTX-M producers. The fosA3 genes were flanked by IS26 and were localized on F2:A�:B� plasmids or on very sim- ilar F33:A�:B� plasmids carrying both blaCTX-M-65 and rmtB. These findings indicate that the fosA3 gene may be coselected by antimicrobials other than fosfomycin. Fosfomycin is a traditional antimicrobial agent with broad-spectrum bactericidal reactivity and good pharmacological properties. It used to be an alternative treatment for uncompli- cated lower urinary tract infections which were caused by a wide variety of bacteria, including Escherichia coli (11, 19, 26). The recent growing prevalence of extended-spectrum �-lacta- mase (ESBL)-producing Enterobacteriaceae and fluoroquinolone- resistant E. coli has rekindled interest in fosfomycin as a therapeu- tic agent in many countries (7, 8, 18). Despite its worldwide use in clinical practice for nearly 4 decades, fosfomycin remains effective against common uropathogens without giving rise to clinically significant resistance (3, 6, 7, 9, 12, 15, 20). The main type of resistance to fosfomycin appears to be chromosome mediated rather than plasmid mediated (17, 21, 23, 27). However, two novel plasmid-mediated fosfomycin-modifying enzymes, FosA3 and FosC2, were recently identified in CTX-M-producing E. coli in Japan (25). Transferable plasmids carrying fosA3 or fosC2 might accelerate the dissemination of fosfomycin resistance around the world. Fosfomycin has been approved for clinical application for many years in China. However, information on the occurrence and characteristics of fosfomycin-resistant E. coli in China is scarce. In the present study, we intended to examine the preva- lence of fosfomycin resistance and plasmid-borne fosfomycin re- sistance genes among E. coli isolates from companion animals. A total of 323 E. coli isolates were recovered from healthy (136 iso- lates) and diseased (187 isolates) pets (248 from dogs and 75 from cats) at 10 pet hospitals in Guangdong Province, China, between 2006 and 2010. The MICs of fosfomycin were determined by the agar dilution method on Mueller-Hinton agar containing 25 �g/ml glucose 6-phosphate, according to guideline M100-S20 of the Clinical and Laboratory Standards Institute (CLSI) (5). Most of the strains (89.9%) studied were susceptible to fosfomycin, whereas 33 iso- lates (10.2%) showed resistance to fosfomycin (MIC � 256 �g/ ml). The 33 isolates were screened for the plasmid-borne fosfomy- cin resistance genes fosA3, fosC2, and fosA by PCR amplification and sequencing with the primers and PCR conditions listed in Table 1. Twenty-nine isolates (9.0%) were positive for fosA3 (Ta- ble 2). No fosC2 or fosA gene was detected among these isolates. Phylogenetic grouping of FosA3 producers as previously de- scribed (4) revealed that these E. coli isolates belonged to three phylogenetic groups (A, B1, and D) (Table 2). Pulsed-field gel electrophoresis (PFGE) (10) was successfully performed on 26 FosA3 producers, and 21 different XbaI PFGE patterns were ob- served. This suggested that the dissemination of fosA3 was not due to the clonal dissemination of fosA3-positive isolates. However, clonal expansion was observed between dogs and cats and be- tween pet hospitals (Table 2). Moreover, three clonally related isolates (HN015, HN7A2, and HN109) which were grouped into phylogenetic group B1 were recovered from different animals and hospitals during 2008 and 2010 (Table 2). Multilocus sequence typing (MLST) analysis revealed that they all belonged to the same sequence type (clonal complex ST448) (data not shown). Received 15 June 2011 Returned for modification 27 September 2011 Accepted 31 December 2011 Published ahead of print 9 January 2012 Address correspondence to Jian-Hua Liu, jhliu@scau.edu.cn. J.H. and X.H. contributed equally to this article. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.05104-11 TABLE 1 Primers and PCR conditions used Primera Sequence (5=–3=) Size (bp) Annealing temp (°C) FosA3-F GCGTCAAGCCTGGCATTT 282 57.5 FosA3-R GCCGTCAGGGTCGAGAAA FosC2-F TGGAGGCTACTTGGATTTG 217 50.5 FosC2-R AGGCTACCGCTATGGATTT IS26-F GCACGCATCACCTCAATACC Unknown 56.7 FosA3-R2 TCATCCAGCGACAAGCACA FosA3-F2 GGGGCTGAGGTATGGAAAGA Unknown 56.1 IS26-R AGGAGATGCTGGCTGAACG FosA-F ATCTGTGGGTCTGCCTGTCGT 271 59.5 FosA-R ATGCCCGCATAGGGCTTCT a Primers were designed in this study. 0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p. 2135–2138 aac.asm.org 2135 o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://dx.doi.org/10.1128/AAC.05104-11 http://aac.asm.org http://aac.asm.org/ MICs of cefotaxime, amikacin, tetracycline, chloramphenicol, and ciprofloxacin were determined by the agar dilution method, and the results were interpreted according to the CLSI breakpoints (5). It revealed that all fosA3-positive isolates were resistant or intermediate to cefotaxime, while 26 and 27 isolates showed resis- tance to amikacin and ciprofloxacin, respectively. The occurrence of rmtB, armA, and blaCTX-M among these fosA3-positive isolates was determined by PCR amplification and sequencing as previ- ously described (2, 22). All of the fosA3-positive isolates were CTX-M producers, and 16 of them produced CTX-M-65 (Table TABLE 2 Characterization of fosA3-carrying Escherichia coli isolates and plasmids Isolatea Date (yr.mo) of isolation Pet hospitalj Originb Resistance phenotypec Resistance gene(s)d Phylogenetic group PFGE typee Plasmid Distance (bp) downstream of fosA3f FAB formulag EcoRI RFLPh Addiction system(s) HN4E2 2008.5 PH1 Dog pharynx CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 1a 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN429 2008.4 PH2 Cat feces* CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 1b 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN2F2 2008.5 PH1 Cat feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB B1 2 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN7A8 2008.1 PH3 Dog feces CTX, AMI, CHL, TET blaCTX-M-65, blaCTX-M-55, rmtB B1 2 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN2B1 2007.12 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB D 3 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN3D12 2008.4 PH3 Cat feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 4 536 F33:A�:B� Ia pemKI, hok-sok, srnBC, ccdAB HN4B5 2008.1 PH3 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A NT 536 F33:A�:B� Ia pemKI, hok-sok, srnBC, ccdAB HN5E3 2008.4 PH1 Dog pus CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 5 536 F33:A�:B� Ib pemKI, hok-sok, srnBC HN4A1 2008.1 PH3 Dog feces* CTX, AMI, CIP, TET blaCTX-M-65, rmtB A 6 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN127 2010.4 PH4 Dog feces* CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB D NT 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN053 2009.7 PH1 Dog feces* CTX, AMI, CIP, TET blaCTX-M-65, rmtB A 7 536 F33:A�:B� Ia pemKI, hok-sok, srnBC HN131 2010.5 PH4 Dog feces CTX, AMI, CIP, TET blaCTX-M-65, rmtB A 8 536 F33:A�:B� Ic pemKI, hok-sok, srnBC HN212 2010.1 PH1 Dog feces CTX, AMI, CIP, TET blaCTX-M-65, rmtB A 9 1,758 F33:A�:B� VI pemKI, srnBC HN1E1 2009.5 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB B1 10 1,758 F2:A�:B� IVc pemKI, hok-sok HN015 2008.1 PH4 Cat feces* CTX, AMI, CIP, TET blaCTX-M-3, rmtB B1 11 1,758 F2:A�:B� ND i pemKI, hok-sok HN7A2 2008.1 PH4 Dog sneeze CTX, AMI, CIP, TET blaCTX-M-3, rmtB B1 11 1,758 F2:A�:B� IVa pemKI, hok-sok HN109 2010.5 PH3 Cat feces CTX, AMI, CIP, TET blaCTX-M-3, rmtB B1 11 1,758 F2:A�:B� ND pemKI, hok-sok HNC50 2006.12 PH1 Dog feces CTX, AMI, CIP, TET blaCTX-M-3, blaCTX-M-65, armA D NT 1,758 F2:A�:B� ND pemKI HNC1 2006.9 PH1 Cat feces CTX, AMI, CIP, TET blaCTX-M-3, rmtB A 12 536 Unknown V pemKI HN225 2010.2 PH1 Cat feces CTX, AMI, CIP, CHL, TET blaCTX-M-27, rmtB D 13 1,758 Unknown ND pemKI, hok-sok HN357 2010.4 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-27, rmtB D 13 1,758 ND ND ND HN1D5 2008.2 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 14 536 F2:A�:B� IIa pemKI, hok-sok HN2E7 2008.5 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-65, rmtB A 15 536 F2:A�:B� IVb pemKI, hok-sok HN3C6 2008.3 PH1 Dog feces CTX, AMI, CIP, TET blaCTX-M-14, rmtB D 16 536 F2:A�:B� IIb pemKI, hok-sok HN5A12 2008.3 PH1 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-27, armA D 17 536 Unknown IIIa pemKI HN061 2009.7 PH5 Dog feces CTX, AMI, CIP, CHL, TET blaCTX-M-14, rmtB D 18 536 ND ND ND HN3B9 2009.4 PH1 Dog feces CTX, CIP, CHL, TET blaCTX-M-65 A 19 370 F2:A�:B� IIIb pemKI, hok-sok HN3E7 2008.2 PH4 Cat feces* CTX, CHL, TET blaCTX-M-3 D 20 370 F2:A�:B� IVb pemKI, hok-sok HN4F8 2008.4 PH3 Dog feces CTX, CIP, CHL, TET blaCTX-M-14 A 21 370 Unknown ND None a Isolates from which the fosA3 gene was transferred to the recipient by conjugation or transformation (isolates HN7A8, HN3D12, HN109, and HN225) are underlined. b Healthy pets are indicated by asterisks. c All isolates and all transconjugants and transformants were resistant to fosfomycin. Resistance phenotypes transferred to the recipient by conjugation are underlined. CTX, cefotaxime; AMK, amikacin; CIP, ciprofloxacin; CHL, chloramphenicol; TET, tetracycline. d Genes that were transferred by conjugation or transformation, as determined by PCR, are underlined. e PFGE types (1, 2, 3, etc.) were assigned by visual inspection of the macrorestriction profile. Patterns that differed by fewer than six bands were considered to represent subtypes within the main group (1a, 1b, etc.). NT, nontypeable. f The size of the spacer region between the 3= end of fosA3 and IS26. g Allele numbers were assigned by submitting the amplicon sequence to the Multilocus Sequence Typing database (www.pubmlst.org/plasmid). h RFLP patterns that differed by only a few bands (1 to 3) were assigned to the same RFLP profile. i ND, not determined. j PH1 to PH5, pet hospitals 1 to 5, respectively. Hou et al. 2136 aac.asm.org Antimicrobial Agents and Chemotherapy o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org http://aac.asm.org/ 2). In addition, 24 and 2 of them carried rmtB and armA, respec- tively. Details for all fosA3-positive isolates are shown in Table 2. Specific primers were designed according to reported surround- ing structures to determine the genetic environment of the fosA3 gene (Table 1). The results showed that all fosA3 genes were flanked by IS26, which was similar to the genetic environment of the first-reported fosA3 gene (25). All fosA3 genes were located 316 bp downstream of IS26. However, the size of the spacer region between the 3= end of fosA3 and IS26 varied (536, 1,758, and 370 bp) (Table 2 and Fig. 1). Moreover, the 1,758-bp region had 79% nucleotide identity with a part of the chromosome sequence of Klebsiella pneumoniae strain 342 and was 100% identical to the sequence downstream of fosA3 in E. coli 08-642, an isolate from Japan (Fig. 1) (25). Conjugation was carried out to determine the transferability of fosA3 genes with E. coli C600 (high level resistance to streptomy- cin) as the recipient (2). Transconjugants were selected on Mac- Conkey agar plates containing fosfomycin (100 �g/ml) and strep- tomycin (2,000 �g/ml) for counterselection. When plasmid cotransfer occurred, a transformation experiment was carried out. Transformants were selected in LB agar plates containing 200 �g/ml fosfomycin by using E. coli DH5� as the recipient. Antimi- crobial susceptibility testing was conducted for transconjugants and transformants, and the transfer of the resistance gene was confirmed by PCR as described above. The fosA3 genes were suc- cessfully transferred to the recipients from 27 donors by conjuga- tion or transformation (Table 2). The 23 transconjugants and 4 transformants all showed extraordinarily high-level resistance to fosfomycin (Table 2). In addition, blaCTX-M and rmtB genes were cotransferred to the recipients with fosA3 from 25 and 18 donors, respectively. Plasmids were assigned to incompatibility groups by PCR-based replicon typing (1). Replicon sequence typing was used to characterize the IncFII plasmids (24). F33:A�:B� and F2:A�:B� were identified in 13 and 10 plasmids carrying fosA3, respectively. F33:A�:B� plasmids also contained blaCTX-M-65 and rmtB and had nearly identical sizes and EcoRI digestion profiles (Table 1 and Fig. 2). Southern blot hybridization was performed on EcoRI digestion fragments of 12 F33:A�:B� plasmids with a digoxigenin-labeled probe specific for fosA3. It showed that fosA3 was located on the same-size band (�15 kb, the largest digestion fragment) in 9 isolates (Fig. 2), demonstrating the presence of an epidemic plasmid responsible for the dissemination of fosA3. However, the predominance of the F33:A�:B� plasmid type was unexpected, since the pets were epidemiologically unrelated and samples had been obtained in different periods at four different hospitals between 2007 and 2010. To better understand the suc- cessful dissemination of these IncFII plasmids carrying fosA3, plasmid addiction systems were determined using primers de- scribed by Mnif et al. (16). pemKI (n � 26), hok-sok (n � 22), and srnBC (n � 13) were the most frequently represented systems, and almost all F33:A�:B� plasmids carried these three addiction sys- tems (Table 2). The occurrence of fosfomycin resistance in E. coli from human and pet animal isolates is still rare in many countries (�5%) (3, 6, 7, 9, 12, 13, 15, 20). However, in this study, a higher prevalence of fosfomycin resistance mainly mediated by FosA3 was observed in E. coli isolates recovered from pets during 2006 and 2010, al- though none of the pets had received fosfomycin treatment. The FIG 1 Comparison of regions flanking fosA3. (I)Escherichia coli 08-642 from Japan (23). (II) The size of the spacer region between the 3= end of fosA3 and IS26 is 1,758 bp (GenBank accession no. JF411006). (III) The size of the spacer region between the 3= end of fosA3 and IS26 is 536 bp (GenBank accession no. JF411007). (IV) The size of the spacer region between the 3= end of fosA3 and IS26 is 370 bp (GenBank accession no. JF411008). FIG 2 Analysis of F33:A�:B� plasmids carrying fosA3. Lanes 1 to 12, HN429, HN7A8, HN2B1, HN3D12, HN4B5, HN4A1, HN127, HN053, HN5E3, HN212, HN4E2, and HN131; lane M, �HindIII and DL15000 marker. (a) Plasmid profiles of transconjugants and transformants carrying F33:A�:B� plasmid. (b) EcoRI restriction digestion profiles of F33:A�:B� plasmids. (c) Southern blot hybridization of EcoRI-digested plasmids with a digoxigenin- labeled fosA3-specific probe. fosA3 in Escherichia coli Isolates from Pets April 2012 Volume 56 Number 4 aac.asm.org 2137 o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org http://aac.asm.org/ association with other resistance determinants has likely favored the dissemination and maintenance of fosA3, since the additional resistance genes, such as blaCTX-M and rmtB, allow coselection of fosA3 by cephalosporins and/or aminoglycosides (especially ami- kacin and gentamicin), which have been frequently used for pet therapy in China (14). In conclusion, the dissemination of the fosA3 gene, which is closely associated with blaCTX-M and rmtB, is mainly driven by horizontal transfer of F33:A�:B� and F2:A�:B� plasmids rather than clonal expansion. Since pets are able to acquire multidrug- resistant pathogens and transmit them to humans due to their close contact, the presence of these resistance bacteria and plas- mids in pets may become a public health concern. Effective anti- microbial policies in veterinary hospitals should be developed in China. Nucleotide sequence accession numbers. The sequences de- termined in this study have been deposited in GenBank under the accession numbers JF411006, JF411007, and JF411008. ACKNOWLEDGMENTS We are grateful to Jun-ichi Wachino for providing the sequence flanking fosA3 for comparison. We thank Minggui Wang for helpful comments on the manuscript. This work was supported in part by grants 30972218 and U1031004 from the National Natural Science Foundation of China. REFERENCES 1. Carattoli A, et al. 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63:219 –228. 2. Chen L, et al. 2007. Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. J. Antimicrob. Chemother. 59:880 – 885. 3. Chislett RJ, White G, Hills T, Turner DP. 2010. Fosfomycin suscepti- bility among extended-spectrum �-lactamase-producing Escherichia coli in Nottingham, UK. J. Antimicrob. Chemother. 65:1076 –1077. 4. Clermont O, Bonacorsi S, Bingen E. 2000. Rapid and simple determina- tion of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555– 4558. 5. Clinical and Laboratory Standards Institute. 2010. Performance stan- dards for antimicrobial susceptibility testing: twentieth informational supplement, M100-S20. CLSI, Wayne, PA. 6. Endimiani A, et al. 2010. In vitro activity of fosfomycin against blaKPC- containing Klebsiella pneumoniae isolates, including those nonsusceptible to tigecycline and/or colistin. Antimicrob. Agents Chemother. 54:526 – 529. 7. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. 2010. Fosfomycin for the treatment of multidrug-resistant, including extended- spectrum �-lactamase producing, Enterobacteriaceae infections: a sys- tematic review. Lancet Infect. Dis. 10:43–50. 8. Falagas ME, Giannopoulou KP, Kokolakis GN, Rafailidis PI. 2008. Fosfomycin: use beyond urinary tract and gastrointestinal infections. Clin. Infect. Dis. 46:1069 –1077. 9. Falagas ME, et al. 2010. Antimicrobial susceptibility of multidrug- resistant (MDR) and extensively drug-resistant (XDR) Enterobacteriaceae isolates to fosfomycin. Int. J. Antimicrob. Agents 35:240 –243. 10. Gautom RK. 1997. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in 1 day. J. Clin. Microbiol. 35:2977–2980. 11. Gupta K, et al. 2011. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the Infectious Diseases Society of America and the Euro- pean Society for Microbiology and Infectious Diseases. Clin. Infect. Dis. 52:e103– e120. 12. Hsu MS, et al. 2011. In vitro susceptibilities of clinical isolates of ertap- enem-non-susceptible Enterobacteriaceae to nemonoxacin, tigecycline, fosfomycin and other antimicrobial agents. Int. J. Antimicrob. Agents. 37:276 –278. 13. Hubka P, Boothe DM. 2011. In vitro susceptibility of canine and feline Escherichia coli to fosfomycin. Vet. Microbiol. 149:277–282. 14. Lei T, et al. 2010. Antimicrobial resistance in Escherichia coli isolates from food animals, animal food products and companion animals in China. Vet. Microbiol. 146:85– 89. 15. Maraki S, et al. 2009. Susceptibility of urinary tract bacteria to fosfomy- cin. Antimicrob. Agents Chemother. 53:4508 – 4510. 16. Mnif B, et al. 2010. Molecular characterization of addiction systems of plasmids encoding extended-spectrum �-lactamases in Escherichia coli. J. Antimicrob. Chemother. 65:1599 –1603. 17. Oteo J, et al. 2009. CTX-M-15-producing urinary Escherichia coli O25b- ST131-phylogroup B2 has acquired resistance to fosfomycin. J. Antimi- crob. Chemother. 64:712–717. 18. Oteo J, Pérez-Vázquez M, Campos J. 2010. Extended-spectrum �-lacta- mase producing Escherichia coli: changing epidemiology and clinical im- pact. Curr. Opin. Infect. Dis. 23:320 –326. 19. Patel SS, Balfour JA, Bryson HM. 1997. Fosfomycin tromethamine. A review of its antibacterial activity, pharmacokinetic properties and thera- peutic efficacy as a single-dose oral treatment for acute uncomplicated lower urinary tract infections. Drugs 53:637– 656. 20. Samonis G, et al. 2010. Antimicrobial susceptibility of Gram-negative nonurinary bacteria to fosfomycin and other antimicrobials. Future Mi- crobiol. 5:961–970. 21. Seoane A, Sangari FJ, Lobo JM. 2010. Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921. Int. J. Antimicrob. Agents 35:413– 414. 22. Sun Y, et al. 2010. High prevalence of blaCTX-M extended-spectrum �-lac- tamase genes in Escherichia coli isolates from pets and emergence of CTX- M-64 in China. Clin. Microbiol. Infect. 16:1475–1481. 23. Takahata S, et al. 2010. Molecular mechanisms of fosfomycin resistance in clinical isolates of Escherichia coli. Int. J. Antimicrob. Agents. 35:333– 337. 24. Villa L, GarcíA-Fernández A, Fortini D, Carattoli A. 2010. Replicon sequence typing of IncF plasmids carrying virulence and resistance deter- minants. J. Antimicrob. Chemother. 65:2518 –2529. 25. Wachino J, Yamane K, Suzuki S, Kimura K, Arakawa Y. 2010. Preva- lence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. Antimicrob. Agents Chemother. 54: 3061–3064. 26. Warren JW, et al. 1999. Guidelines for antimicrobial treatment of un- complicated acute bacterial cystitis and acute pyelonephritis in women. Clin. Infect. Dis. 29:745–758. 27. Xu H, Miao V, Kwong W, Xia R, Davies J. 2011. Identification of a novel fosfomycin resistance gene (fosA2) in Enterobacter cloacae from the Salmon River, Canada. Lett. Appl. Microbiol. 52:427– 429. Hou et al. 2138 aac.asm.org Antimicrobial Agents and Chemotherapy o n A p ril 5 , 2 0 2 1 a t C A R N E G IE M E L L O N U N IV L IB R h ttp ://a a c.a sm .o rg / D o w n lo a d e d fro m http://aac.asm.org http://aac.asm.org/