- Email: [email protected]
Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921
Letters to the Editor / International Journal of Antimicrobial Agents 35 (2010) 410–416 [6] Stepanova MN, Pimkin M, Nikulin AA, Kozyreva VK, Agapova ED, Edelstein MV. Convergent in vivo and in vitro selection of ceftazidime resistance mutations at position 167 of CTX-M-3 -lactamase in hypermutable Escherichia coli strains. Antimicrob Agents Chemother 2008;52:1297–301. [7] Bae IK, Lee BH, Hwang HY, Jeong SH, Hong SG, Chang CL, et al. A novel ceftazidime-hydrolysing extended-spectrum -lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop. J Antimicrob Chemother 2006;58:315–9.
Guo-Bao Tian a,b Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA b Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, School of Life Sciences, Sichuan University, Chengdu, China
a
Jennifer M. Adams-Haduch Zubair A. Qureshi Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA Hong-Ning Wang Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, School of Life Sciences, Sichuan University, Chengdu, China Yohei Doi ∗ Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA
a
∗ Corresponding
author. Tel.: +1 412 648 9445; fax: +1 412 648 8521. E-mail address: [email protected] (Y. Doi) 11 November 2009 15 December 2009
doi:10.1016/j.ijantimicag.2009.12.004
Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921 Sir, Fosfomycin is a cell wall-active antibiotic introduced into clinical practice around 1970 [1]. Despite its broad spectrum of activity and good pharmacological properties, its use has been somewhat hampered by the high number of spontaneous resistant mutants isolated following antibiotic challenge in many bacterial pathogens [2], most of them carrying chromosomal mutations impairing drug transport.
413
Fosfomycin has been used steadily for the treatment of lower urinary tract infections. Recently, the emergence of resistance to newer antibiotics has resulted in a renewed interest in fosfomycin as a therapeutic agent [3]. The existence of plasmids conferring resistance to fosfomycin was demonstrated in strains of Serratia marcescens isolated in the ˜ Hospital ‘Nuestra Senora de Covadonga’ (Asturias, Spain) [4]. The fosfomycin resistance gene fosA in some of those plasmids was found to reside on a transposable element called Tn2921 [5]. The fosA gene was shown to encode a fosfomycin-inactivating mechanism [6] that consisted of enzymatic coupling with glutathione. The transposable element Tn2921 was estimated to be ca. 12.5 kb long, but its composition was unknown except for the presence of the fosA gene and the two terminal copies of insertion sequence IS2921, which are closely related to IS10 (1.3 kb each) [7]. To gain further insight into the gene content of Tn2921 and the possible mechanisms leading to the assembly of the transposon, the complete nucleotide sequence of Tn2921 in plasmid pSU912 was determined. The transposon was flanked by identical 1329-bp long insertion sequences, with only 1 bp difference from IS10-right (IS10R) and 14 bp with IS10-left (IS10L). A single sequence of 12 452 bp containing the complete 12 434 bp of the transposon and the two target 9-bp direct repeats has been deposited in GenBank (accession no. FJ829469). The numbering used in the text makes reference to this GenBank entry. The first open-reading frame (ORF) next to IS10L (1347–1701) contains a 120-amino acid N-terminal fragment of a tryptophanyltRNA synthetase II truncated by the insertion of IS10. The 9-mer Tn10 target site sequence GGCGAAGCC (1339–1347) signals the limit of the gene remnant and indicates that IS10 was integrated here by transposition. The fosA gene (1901–2326) is the fosfomycin resistance gene. Genes similar to fosA are relatively frequent in the chromosomes of many bacteria, where they play functions related or not with antibiotic resistance. Protein BLAST searches identified three complete ORFs in the region (2312–6033) contiguous to fosA. The first ORF encoded a transcriptional regulator belonging to the LacI family. The next encoded a conserved hypothetical protein with a possible function as a glycoside hydrolase. The final ORF encodes a sugar transporter belonging to the major facilitator superfamily (MFS). This gene arrangement is suggestive of a system operating to sense, transport and hydrolyse some sugar molecules for further use in bacterial metabolism. The next two ORFs in Tn2921 (6068–8172) form part of a 5methylcytosine GTP-dependent restriction system. The mcrC gene is complete, whilst the mcrB gene is 5 truncated and is probably not
Fig. 1. Gene map of transposon Tn2921. Genes that are inactive due to truncation are filled with dots. The fosA gene is in black. The lower part of the figure shows the segment from the Enterobacter cancerogenus genome that is homologous and co-linear with the left half of the transposon (grey blocks). Deletion of gene ENTCAN 00725 and insertion of IS10L at the indicated point (black arrowhead) of ENTCAN 00726 could be the origin of the transposon. Segments from Tn3 and Tn7 are also indicated.
414
Letters to the Editor / International Journal of Antimicrobial Agents 35 (2010) 410–416
functional, rendering the mcrC gene useless because both proteins are required for function. The sequence from here to the right end of the transposon consists of nested remnants of transposable elements following a ‘transposon in transposon’ design type. Next to mcrB (8172–9872), a non-functional fragment of the Tn3 transposase gene was identified. The sequence from 9874 to 11 114 corresponded to the left end of the transposon Tn7 including the left IR and the carboxylterminal end of the site-specific recombinase IntI2. The presence of the left IR of the transposon Tn7 suggests that Tn7 could have transposed into Tn3. Tn7 was interrupted by integration of IS10R, which constitutes the right end of Tn2921 (Fig. 1). The gene order observed in the central region of Tn2921 was not found in any other sequence deposited in the public sequence databases. However, the 5.6 kb DNA region between IS10L and the mcr genes, including fosA (1339–5967), showed 90% identity with a genomic segment from Enterobacter cancerogenus ATCC 33516 (GenBank accession no. ABWM00000000). A chromosomal region like this could be the origin of the transposon. The deletion of a gene (ENTCAN 00725) and integration of IS10 into ENTCAN 00726 will exactly produce the left half of Tn2921 (Fig. 1). In conclusion, Tn2921 provides a good example of evolution of an antibiotic resistance composite transposon. We can assume that an enterobacterial gene encoding a protein of the glyoxalase family was deregulated and subsequently incorporated as a fosfomycin resistance gene in a transposable element by successive integration of two identical ISs. Furthermore, the presence of genes unrelated to resistance and of many gene remnants in the central part of Tn2921 suggests that this transposon may have evolved recently under the selective pressure of fosfomycin usage. Funding: This work was supported by grant PI05/0894 from ‘Fondo de investigación Sanitaria’ of the Spanish Ministry of Science and Innovation. Competing interests: None declared. Ethical approval: Not required. References [1] Hendlin D, Stapley EO, Jackson M, Wallick H, Miller AK, Wolf FJ, et al. Phosphonomycin, a new antibiotic produced by strains of Streptomyces. Science 1969;166:122–3. [2] Dulaney EL, Ruby CL. In vitro development of resistance to fosfomycin. J Antibiot (Tokyo) 1977;30:252–61. [3] Roussos N, Karageorgopoulos DE, Samonis G, Falagas ME. Clinical significance of the pharmacokinetic and pharmacodynamic characteristics of fosfomycin for the treatment of patients with systemic infections. Int J Antimicrob Agents 2009;34:506–15. [4] Mendoza C, Garcia JM, Llaneza J, Mendez FJ, Hardisson C, Ortiz JM. Plasmiddetermined resistance to fosfomycin in Serratia marcescens. Antimicrob Agents Chemother 1980;18:215–9. [5] García-Lobo JM, Ortiz JM. Tn2921, a transposon encoding fosfomycin resistance. J Bacteriol 1982;151:477–9. [6] León J, García-Lobo JM, Navas J, Ortiz JM. Fosfomycin-resistance plasmids determine an intracellular modification of fosfomycin. J Gen Microbiol 1985;131:1649–55. [7] Navas J, García-Lobo JM, León J, Ortíz JM. Structural and functional analyses of the fosfomycin resistance transposon Tn2921. J Bacteriol 1985;162:1061–7.
Asunción Seoane Felix J. Sangari Juan M. García Lobo ∗ Instituto de Biomedicina y Biotecnología de Cantabria (UC-IDICAN-CSIC), and Departamento de Biología Molecular, Universidad de Cantabria, Cardenal Herrera Oria s/n, 39011 Santander, Spain ∗ Corresponding
author. Tel.: +34 942 201 948; fax: +34 942 201 945. E-mail address: [email protected] (J.M. García Lobo) doi:10.1016/j.ijantimicag.2009.12.006
Activity of tigecycline against meticillin-resistant Staphylococcus aureus (MRSA) from respiratory tract sources Sir, Meticillin-resistant Staphylococcus aureus (MRSA) is a recognised causative agent of community-acquired pneumonia (CAP) whose incidence and resistance to antibiotics has been increasing recently [1]. Tigecycline is approved in the USA for treatment of MRSA in complicated intra-abdominal and complicated skin and skin-structure infections and was recently approved for the treatment of CAP in 2008, although the latter indication does not include MRSA [2]. The Tigecycline Evaluation Surveillance Trial (T.E.S.T.) monitors the global in vitro activity of tigecycline compared with several other agents. This study summarises the in vitro activity of tigecycline and appropriate comparators against MRSA from community-associated (CA) and hospital-associated (HA) respiratory tract infections (RTIs). Clinical respiratory isolates were collected from T.E.S.T. from January 2004 to March 2009. Only one isolate per patient was collected. Isolates were from respiratory sources including trachea, sputum, sinuses, lungs and bronchial tissue. Minimum inhibitory concentrations (MICs) were determined using Sensititre® panels (Trek Diagnostic Systems, Westlake, OH) in line with the Clinical and Laboratory Standards Institute (CLSI) recommended broth microdilution testing method [3]. Breakpoints used were as defined by the CLSI [4] or by the USA Food and Drug Administration (FDA) [2] for tigecycline. A total of 1285 respiratory MRSA isolates were collected from patients with HA-RTIs (1194 isolates) or CA-RTIs (91 isolates). Table 1 shows the susceptibilities of MRSA from CA-RTIs and HA-RTIs to tigecycline and three comparator agents. The MIC ranges for tigecycline, linezolid, minocycline and vancomycin against the 1285 isolates were ≤0.008–0.5, ≤0.5–4, ≤0.25 to >8 and ≤0.12–2 g/mL, respectively. Overall, MIC ranges for each drug against CA and HA isolates were similar, with the exception of minocycline whose MIC range against CA isolates was ≤0.25–4 g/mL but was higher against HA isolates with a MIC range of ≤0.25 to >8 g/mL (Table 1). In terms of MIC90 (MIC for 90% of the organisms), all agents with the exception of minocycline exhibited similar MIC90 values for CA-MRSA and HA-MRSA isolates. For example, tigecycline was the most active agent and exhibited a MIC90 of 0.25 g/mL against both HA and CA isolates (Table 1). Similarly, linezolid exhibited a MIC90 of 4 g/mL and vancomycin exhibited a MIC90 of 1 g/mL against both HA and CA isolates (Table 1). Minocycline exhibited a MIC90 similar to tigecycline against CA isolates (0.5 g/mL) but a MIC90 of 4 g/mL against HA isolates, reflective of the higher MIC range of this agent against HA isolates (Table 1). Tigecycline, linezolid and vancomycin all exhibited susceptibilities of 100% against all MRSA without regard to source. By contrast, although 100% of CA isolates were susceptible to minocycline, susceptibility of HA isolates was 94% (P = 0.0080) (Table 1). Although linezolid and vancomycin are clinically proven agents against MRSA, in the present study tigecycline has demonstrated substantial in vitro activity against the same respiratory MRSA, including isolates that were resistant to minocycline. The ability of S. aureus to survive within host cells has been reported to contribute to their pathogenicity, persistence and ability to cause recurrent infections [5]. Therapeutic failures may in part be due to the inability of certain antibiotics to reach intracellularly localised S. aureus. Hence, antibiotics with intracellular activity may play an important role in treating such infections. Tigecycline has been shown to achieve high intracellular concentrations whilst displaying intracellular bacteriostatic activity against S. aureus [6]. This report, taken together with data from the present study, may support the
Guo-Bao Tian a,b Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA b Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, School of Life Sciences, Sichuan University, Chengdu, China
a
Jennifer M. Adams-Haduch Zubair A. Qureshi Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA Hong-Ning Wang Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, School of Life Sciences, Sichuan University, Chengdu, China Yohei Doi ∗ Division of Infectious Diseases, University of Pittsburgh Medical Center, S829 Scaife Hall, 3550 Terrace Street, Pittsburgh, PA, USA
a
∗ Corresponding
author. Tel.: +1 412 648 9445; fax: +1 412 648 8521. E-mail address: [email protected] (Y. Doi) 11 November 2009 15 December 2009
doi:10.1016/j.ijantimicag.2009.12.004
Complete nucleotide sequence of the fosfomycin resistance transposon Tn2921 Sir, Fosfomycin is a cell wall-active antibiotic introduced into clinical practice around 1970 [1]. Despite its broad spectrum of activity and good pharmacological properties, its use has been somewhat hampered by the high number of spontaneous resistant mutants isolated following antibiotic challenge in many bacterial pathogens [2], most of them carrying chromosomal mutations impairing drug transport.
413
Fosfomycin has been used steadily for the treatment of lower urinary tract infections. Recently, the emergence of resistance to newer antibiotics has resulted in a renewed interest in fosfomycin as a therapeutic agent [3]. The existence of plasmids conferring resistance to fosfomycin was demonstrated in strains of Serratia marcescens isolated in the ˜ Hospital ‘Nuestra Senora de Covadonga’ (Asturias, Spain) [4]. The fosfomycin resistance gene fosA in some of those plasmids was found to reside on a transposable element called Tn2921 [5]. The fosA gene was shown to encode a fosfomycin-inactivating mechanism [6] that consisted of enzymatic coupling with glutathione. The transposable element Tn2921 was estimated to be ca. 12.5 kb long, but its composition was unknown except for the presence of the fosA gene and the two terminal copies of insertion sequence IS2921, which are closely related to IS10 (1.3 kb each) [7]. To gain further insight into the gene content of Tn2921 and the possible mechanisms leading to the assembly of the transposon, the complete nucleotide sequence of Tn2921 in plasmid pSU912 was determined. The transposon was flanked by identical 1329-bp long insertion sequences, with only 1 bp difference from IS10-right (IS10R) and 14 bp with IS10-left (IS10L). A single sequence of 12 452 bp containing the complete 12 434 bp of the transposon and the two target 9-bp direct repeats has been deposited in GenBank (accession no. FJ829469). The numbering used in the text makes reference to this GenBank entry. The first open-reading frame (ORF) next to IS10L (1347–1701) contains a 120-amino acid N-terminal fragment of a tryptophanyltRNA synthetase II truncated by the insertion of IS10. The 9-mer Tn10 target site sequence GGCGAAGCC (1339–1347) signals the limit of the gene remnant and indicates that IS10 was integrated here by transposition. The fosA gene (1901–2326) is the fosfomycin resistance gene. Genes similar to fosA are relatively frequent in the chromosomes of many bacteria, where they play functions related or not with antibiotic resistance. Protein BLAST searches identified three complete ORFs in the region (2312–6033) contiguous to fosA. The first ORF encoded a transcriptional regulator belonging to the LacI family. The next encoded a conserved hypothetical protein with a possible function as a glycoside hydrolase. The final ORF encodes a sugar transporter belonging to the major facilitator superfamily (MFS). This gene arrangement is suggestive of a system operating to sense, transport and hydrolyse some sugar molecules for further use in bacterial metabolism. The next two ORFs in Tn2921 (6068–8172) form part of a 5methylcytosine GTP-dependent restriction system. The mcrC gene is complete, whilst the mcrB gene is 5 truncated and is probably not
Fig. 1. Gene map of transposon Tn2921. Genes that are inactive due to truncation are filled with dots. The fosA gene is in black. The lower part of the figure shows the segment from the Enterobacter cancerogenus genome that is homologous and co-linear with the left half of the transposon (grey blocks). Deletion of gene ENTCAN 00725 and insertion of IS10L at the indicated point (black arrowhead) of ENTCAN 00726 could be the origin of the transposon. Segments from Tn3 and Tn7 are also indicated.
414
Letters to the Editor / International Journal of Antimicrobial Agents 35 (2010) 410–416
functional, rendering the mcrC gene useless because both proteins are required for function. The sequence from here to the right end of the transposon consists of nested remnants of transposable elements following a ‘transposon in transposon’ design type. Next to mcrB (8172–9872), a non-functional fragment of the Tn3 transposase gene was identified. The sequence from 9874 to 11 114 corresponded to the left end of the transposon Tn7 including the left IR and the carboxylterminal end of the site-specific recombinase IntI2. The presence of the left IR of the transposon Tn7 suggests that Tn7 could have transposed into Tn3. Tn7 was interrupted by integration of IS10R, which constitutes the right end of Tn2921 (Fig. 1). The gene order observed in the central region of Tn2921 was not found in any other sequence deposited in the public sequence databases. However, the 5.6 kb DNA region between IS10L and the mcr genes, including fosA (1339–5967), showed 90% identity with a genomic segment from Enterobacter cancerogenus ATCC 33516 (GenBank accession no. ABWM00000000). A chromosomal region like this could be the origin of the transposon. The deletion of a gene (ENTCAN 00725) and integration of IS10 into ENTCAN 00726 will exactly produce the left half of Tn2921 (Fig. 1). In conclusion, Tn2921 provides a good example of evolution of an antibiotic resistance composite transposon. We can assume that an enterobacterial gene encoding a protein of the glyoxalase family was deregulated and subsequently incorporated as a fosfomycin resistance gene in a transposable element by successive integration of two identical ISs. Furthermore, the presence of genes unrelated to resistance and of many gene remnants in the central part of Tn2921 suggests that this transposon may have evolved recently under the selective pressure of fosfomycin usage. Funding: This work was supported by grant PI05/0894 from ‘Fondo de investigación Sanitaria’ of the Spanish Ministry of Science and Innovation. Competing interests: None declared. Ethical approval: Not required. References [1] Hendlin D, Stapley EO, Jackson M, Wallick H, Miller AK, Wolf FJ, et al. Phosphonomycin, a new antibiotic produced by strains of Streptomyces. Science 1969;166:122–3. [2] Dulaney EL, Ruby CL. In vitro development of resistance to fosfomycin. J Antibiot (Tokyo) 1977;30:252–61. [3] Roussos N, Karageorgopoulos DE, Samonis G, Falagas ME. Clinical significance of the pharmacokinetic and pharmacodynamic characteristics of fosfomycin for the treatment of patients with systemic infections. Int J Antimicrob Agents 2009;34:506–15. [4] Mendoza C, Garcia JM, Llaneza J, Mendez FJ, Hardisson C, Ortiz JM. Plasmiddetermined resistance to fosfomycin in Serratia marcescens. Antimicrob Agents Chemother 1980;18:215–9. [5] García-Lobo JM, Ortiz JM. Tn2921, a transposon encoding fosfomycin resistance. J Bacteriol 1982;151:477–9. [6] León J, García-Lobo JM, Navas J, Ortiz JM. Fosfomycin-resistance plasmids determine an intracellular modification of fosfomycin. J Gen Microbiol 1985;131:1649–55. [7] Navas J, García-Lobo JM, León J, Ortíz JM. Structural and functional analyses of the fosfomycin resistance transposon Tn2921. J Bacteriol 1985;162:1061–7.
Asunción Seoane Felix J. Sangari Juan M. García Lobo ∗ Instituto de Biomedicina y Biotecnología de Cantabria (UC-IDICAN-CSIC), and Departamento de Biología Molecular, Universidad de Cantabria, Cardenal Herrera Oria s/n, 39011 Santander, Spain ∗ Corresponding
author. Tel.: +34 942 201 948; fax: +34 942 201 945. E-mail address: [email protected] (J.M. García Lobo) doi:10.1016/j.ijantimicag.2009.12.006
Activity of tigecycline against meticillin-resistant Staphylococcus aureus (MRSA) from respiratory tract sources Sir, Meticillin-resistant Staphylococcus aureus (MRSA) is a recognised causative agent of community-acquired pneumonia (CAP) whose incidence and resistance to antibiotics has been increasing recently [1]. Tigecycline is approved in the USA for treatment of MRSA in complicated intra-abdominal and complicated skin and skin-structure infections and was recently approved for the treatment of CAP in 2008, although the latter indication does not include MRSA [2]. The Tigecycline Evaluation Surveillance Trial (T.E.S.T.) monitors the global in vitro activity of tigecycline compared with several other agents. This study summarises the in vitro activity of tigecycline and appropriate comparators against MRSA from community-associated (CA) and hospital-associated (HA) respiratory tract infections (RTIs). Clinical respiratory isolates were collected from T.E.S.T. from January 2004 to March 2009. Only one isolate per patient was collected. Isolates were from respiratory sources including trachea, sputum, sinuses, lungs and bronchial tissue. Minimum inhibitory concentrations (MICs) were determined using Sensititre® panels (Trek Diagnostic Systems, Westlake, OH) in line with the Clinical and Laboratory Standards Institute (CLSI) recommended broth microdilution testing method [3]. Breakpoints used were as defined by the CLSI [4] or by the USA Food and Drug Administration (FDA) [2] for tigecycline. A total of 1285 respiratory MRSA isolates were collected from patients with HA-RTIs (1194 isolates) or CA-RTIs (91 isolates). Table 1 shows the susceptibilities of MRSA from CA-RTIs and HA-RTIs to tigecycline and three comparator agents. The MIC ranges for tigecycline, linezolid, minocycline and vancomycin against the 1285 isolates were ≤0.008–0.5, ≤0.5–4, ≤0.25 to >8 and ≤0.12–2 g/mL, respectively. Overall, MIC ranges for each drug against CA and HA isolates were similar, with the exception of minocycline whose MIC range against CA isolates was ≤0.25–4 g/mL but was higher against HA isolates with a MIC range of ≤0.25 to >8 g/mL (Table 1). In terms of MIC90 (MIC for 90% of the organisms), all agents with the exception of minocycline exhibited similar MIC90 values for CA-MRSA and HA-MRSA isolates. For example, tigecycline was the most active agent and exhibited a MIC90 of 0.25 g/mL against both HA and CA isolates (Table 1). Similarly, linezolid exhibited a MIC90 of 4 g/mL and vancomycin exhibited a MIC90 of 1 g/mL against both HA and CA isolates (Table 1). Minocycline exhibited a MIC90 similar to tigecycline against CA isolates (0.5 g/mL) but a MIC90 of 4 g/mL against HA isolates, reflective of the higher MIC range of this agent against HA isolates (Table 1). Tigecycline, linezolid and vancomycin all exhibited susceptibilities of 100% against all MRSA without regard to source. By contrast, although 100% of CA isolates were susceptible to minocycline, susceptibility of HA isolates was 94% (P = 0.0080) (Table 1). Although linezolid and vancomycin are clinically proven agents against MRSA, in the present study tigecycline has demonstrated substantial in vitro activity against the same respiratory MRSA, including isolates that were resistant to minocycline. The ability of S. aureus to survive within host cells has been reported to contribute to their pathogenicity, persistence and ability to cause recurrent infections [5]. Therapeutic failures may in part be due to the inability of certain antibiotics to reach intracellularly localised S. aureus. Hence, antibiotics with intracellular activity may play an important role in treating such infections. Tigecycline has been shown to achieve high intracellular concentrations whilst displaying intracellular bacteriostatic activity against S. aureus [6]. This report, taken together with data from the present study, may support the