New Quinolone Resistance Phenomenon in Salmonella enterica: Nalidixic Acid-Susceptible Isolates with Reduced Fluoroquinolone Susceptibility
http://www.100md.com
微生物临床杂志 2005年第11期
Antimicrobial Research Laboratory
Laboratory of Human Microbial Ecology
Enteric Bacteria Laboratory, Department of Bacterial and Inflammatory Diseases, National Public Health Institute, Turku and Helsinki
Department of Medicine, Turku University Hospital, Turku, Finland
ABSTRACT
We describe the emergence of a new quinolone resistance pattern in Salmonella enterica isolates from Southeast Asia. These isolates are susceptible to nalidixic acid but exhibit reduced susceptibility to ciprofloxacin. The increase of such strains may threaten the value of the nalidixic acid disk test to screen for reduced fluoroquinolone susceptibility in salmonellas.
TEXT
The fluoroquinolone susceptibility of Salmonella enterica isolates has been surveyed in the National Public Health Institute, Turku and Helsinki, Finland, since 1995 by analyzing 100 to 200 isolates yearly collected from Finnish travelers returning from abroad. Between 1995 and 1999, the annual proportion of reduced fluoroquinolone susceptibility increased significantly among all foreign isolates (6, 7). The increase was especially striking among isolates from travelers returning from Southeast Asia (6). From 1995 to 2002, all Salmonella isolates studied which showed reduced ciprofloxacin susceptibility were uniformly resistant to nalidixic acid. Based on these findings, we have validated the use of the nalidixic acid screening test in the detection of decreased fluoroquinolone susceptibility in salmonellas (5). Among 1,010 Salmonella isolates included in that study, identification of nalidixic acid resistance by the disk diffusion method provided a sensitivity of 100% and a specificity of 87.3% to screen for isolates with reduced susceptibility (5). In 2004, the use of the nalidixic acid screening test was recommended by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) for the detection of invasive Salmonella isolates (14).
During our annual survey of fluoroquinolone susceptibility of Salmonella isolates in 2003, we focused attention on the emergence of a new quinolone resistance pattern among salmonellas originating from Southeast Asia. These isolates showed reduced ciprofloxacin susceptibility but were susceptible to nalidixic acid.
Susceptibility testing. The MICs of the isolates to ciprofloxacin and to nalidixic acid were determined by an Etest according to the manufacturer's instructions. In addition, the susceptibilities to these antimicrobials were determined by the disk diffusion test (13). Mueller-Hinton II agar (Becton Dickinson and Company, Sparks, MD) was used as the culture medium. Etests were purchased from AB Biodisk, Solna, Sweden, and the antimicrobial disks were from Oxoid Ltd. (Oxoid AB, Sollentuna, Sweden). The MIC breakpoints used for susceptibility and resistance to ciprofloxacin and nalidixic acid were 1 and 4 μg/ml and 8 and 32 μg/ml, respectively, as recommended by the Clinical and Laboratory Standards Institute (3). The corresponding inhibition zone diameters of the disk diffusion test for nalidixic acid were 19 and 13 mm (3). The MIC breakpoint value used for reduced ciprofloxacin susceptibility was 0.125 μg/ml. All measurements were repeated three times.
Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were used as controls in susceptibility testing. Also, three local Salmonella enterica strains were used as controls, one fully ciprofloxacin susceptible, one with reduced ciprofloxacin susceptibility, and one highly ciprofloxacin resistant.
Serotyping of the isolates was performed in the Enteric Bacteria Laboratory of the National Public Health Institute, Helsinki, Finland.
Pyrosequencing was applied to detect point mutations at the positions Ser83 and Asp87 in the quinolone resistance-determining region (QRDR) of the gyrA gene in salmonellas (20). Pyrosequencing was performed by using a PSQ 96 sample preparation kit, streptavidin-coated Sepharose beads (Amersham Biosciences, Piscataway, N.J.), a vacuum prep workstation, and a PSQ 96MA instrument (Biotage AB, Uppsala, Sweden) according to the instructions of the manufacturer. In one reaction, 20 μl of the PCR product and 15 pmol of a suitable sequencing primer were used (Table 1). The full details of the technique are available at http://www.pyrosequencing.com.
Characteristics of Salmonella isolates. During 2003 and 2004, we identified a total of 16 isolates showing a novel resistance pattern (Fig. 1 and Table 2). All of these isolates showed reduced ciprofloxacin susceptibility, with the MICs ranging between 0.125 and 0.5 μg/ml. The MICs of nalidixic acid varied from 4 to 16 μg/ml. Eight isolates were fully susceptible, and four isolates were intermediately resistant to nalidixic acid in all three measurements. For four isolates, the MICs varied between susceptible and intermediately resistant. The disk diffusion test results for nalidixic acid varied between susceptible and intermediately resistant. For two isolates, the inhibition zone diameter was 13 mm in one of three measurements.
All isolates were recovered from stool. Twelve isolates were from travelers returning from Thailand, and four isolates were from travelers returning from Malaysia. The isolates represented five different serotypes: Corvallis (n = 7), Stanley (n = 6), Mbandaka (n = 1), Montevideo (n = 1), and Virginia (n = 1). None of the isolates had a mutation at codon Ser83 or Asp87 in the QRDR of the gyrA gene.
These 16 isolates comprised 16.8% of the total 95 Salmonella isolates and 31.4% of the 51 isolates with reduced ciprofloxacin susceptibility recovered during the study period from patients returning from Thailand and Malaysia.
We describe here the emergence of a novel quinolone resistance pattern among Salmonella isolates originating from Southeast Asia. A population with such a resistance pattern has not been previously described. Anecdotal reports have been published on other enterobacteria showing similar resistance characteristics, but such strains have been solitary, as well as rare (12).
The existence and plausible future increase of Salmonella strains that are susceptible to nalidixic acid but show reduced susceptibility to ciprofloxacin may impede the detection of quinolone resistance of salmonellas in areas where such strains are common, since the nalidixic acid disk diffusion test may no longer be valid as a tool to screen for reduced fluoroquinolone susceptibility.
The mechanism of resistance in the Salmonella isolates described here is still unknown. The presence of five different serotypes among these non-fluoroquinolone-susceptible isolates excludes the possibility of the spread of only one clone. The absence of mutations at codon Ser83 or Asp87 of the QRDR of gyrA, which are the two most common sites of mutation leading to quinolone resistance (1, 4, 5, 6, 17, 19), suggests the involvement of other resistance mechanisms. The isolates were not characterized for other mutations; thus, mutations at other locations of the QRDR remain possible. Another plausible explanation for this type of resistance involves the presence of efflux pump mechanisms. It has been shown that exposure to low concentrations of fluoroquinolones may lead to activation of the efflux pump system and a reduction in susceptibility, even when there are no mutations in gyrA (2, 18). Moreover, a possible role of plasmid-mediated resistance (9) in these isolates must be kept in mind, considering that this resistance pattern was observed in multiple serovars.
We believe that the detection of low-level ciprofloxacin resistance in salmonellas is important for epidemiological, as well as for clinical, purposes. Although nontyphoidal Salmonella strains characteristically cause gastroenteritis, they can sometimes produce an invasive infection leading to serious complications. If such an infection is caused by a non-fluoroquinolone-susceptible Salmonella strain, treatment with another antimicrobial group may be indicated. Based on several reports, the use of fluoroquinolones in infections caused by Salmonella isolates with reduced susceptibility may lead to treatment failures (1, 8, 10, 11, 15-17, 21).
The Salmonella population described here may prove problematic, especially in those clinical laboratories that use the nalidixic acid disk diffusion test to screen for reduced ciprofloxacin susceptibility in salmonellas. It is recommended that at least invasive Salmonella isolates from travelers returning from Southeast Asia be tested by a fluoroquinolone Etest or another suitable MIC method.
Nucleotide sequence accession numbers. The nucleotide sequences determined in this study have been deposited in the EMBL database under accession numbers AM076385 to AM076400.
REFERENCES
Brown, J. C., C. J. Thomson, and S. G. Amyes. 1996. Mutations of the gyrA gene of clinical isolates of Salmonella typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quinolone drugs. J. Antimicrob. Chemother. 37:351-356.
Cebrián, L., J. C. Rodríguez, I. Escribano, and G. Royo. 2005. Characterization of Salmonella spp. mutants with reduced fluoroquinolone susceptibility: importance of efflux pump mechanisms. Chemotherapy 51:40-43.
Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing. Fifteenth informational supplement M100-S15. Clinical and Laboratory Standards Institute, Wayne, Pa.
Griggs, D. J., K. Gensberg, and L. J. Piddock. 1996. Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals. Antimicrob. Agents Chemother. 40:1009-1013.
Hakanen, A., P. Kotilainen, J. Jalava, A. Siitonen, and P. Huovinen. 1999. Detection of decreased fluoroquinolone susceptibility in salmonellas and validation of nalidixic acid screening test. J. Clin. Microbiol. 37:3572-3577.
Hakanen, A., P. Kotilainen, P. Huovinen, H. Helenius, and A. Siitonen. 2001. Reduced fluoroquinolone susceptibility in Salmonella enterica serotypes in travelers returning from Southeast Asia. Emerg. Infect. Dis. 7:996-1003.
Hakanen, A., A. Siitonen, P. Kotilainen, and P. Huovinen. 1999. Increasing fluoroquinolone resistance in salmonella serotypes in Finland during 1995-1997. J. Antimicrob. Chemother. 43:145-148.
Helms, M., P. Vastrup, P. Gerner-Smidt, and K. Mlbak. 2002. Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium. Emerg. Infect. Dis. 8:490-495.
Jacoby, G. A. 2005. Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41:120S-126S.
McCarron, B., and W. C. Love. 1997. Acalculous nontyphoidal salmonellal cholecystitis requiring surgical intervention despite ciprofloxacin therapy: report of three cases. Clin. Infect. Dis. 24:707-709.
Mlbak, K., D. L. Baggesen, F. M. Aarestrup, J. M. Ebbesen, J. Engberg, K. Frydendahl, P. Gerner-Smidt, A. M. Petersen, and H. C. Wegener. 1999. An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype Typhimurium DT104. N. Engl. J. Med. 341:1420-1425.
Moniot-Ville, N., J. Guibert, N. Moreau, J. F. Acar, E. Collatz, and L. Gutmann. 1991. Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid. Antimicrob. Agents Chemother. 35:519-523.
National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial disk susceptibility tests, 8th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement M100-S14. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Ouabdesselam, S., J. Tankovic, and C. J. Soussy. 1996. Quinolone resistance mutations in the gyrA gene of clinical isolates of Salmonella. Microb. Drug. Resist. 2:299-302.
Pers, C., P. Sogaard, and L. Pallesen. 1996. Selection of multiple resistance in Salmonella enteritidis during treatment with ciprofloxacin. Scand. J. Infect. Dis. 28:529-531.
Piddock, L. J., D. J. Griggs, M. C. Hall, and Y. F. Jin. 1993. Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients. Antimicrob. Agents Chemother. 37:662-666.
Piddock, L. J., Y. F. Jin, M. A. Webber, and M. J. Everett. 2002. Novel ciprofloxacin-resistant, nalidixic acid-susceptible mutant of Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2276-2278.
Reyna, F., M. Huesca, V. Gonzalez, and L. Y. Fuchs. 1995. Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance. Antimicrob. Agents Chemother. 39:1621-1623.
Ronaghi, M., M. Uhlen, and P. Nyren. 1998. A sequencing method based on real-time pyrophosphate. Science 281:363, 365.
Vasallo, F. J., P. Martin-Rabadan, L. Alcala, J. M. Garcia-Lechuz, M. Rodriguez-Creixems, and E. Bouza. 1998. Failure of ciprofloxacin therapy for invasive nontyphoidal salmonellosis. Clin. Infect. Dis. 26:535-536.(Antti J. Hakanen, Mariann)
Laboratory of Human Microbial Ecology
Enteric Bacteria Laboratory, Department of Bacterial and Inflammatory Diseases, National Public Health Institute, Turku and Helsinki
Department of Medicine, Turku University Hospital, Turku, Finland
ABSTRACT
We describe the emergence of a new quinolone resistance pattern in Salmonella enterica isolates from Southeast Asia. These isolates are susceptible to nalidixic acid but exhibit reduced susceptibility to ciprofloxacin. The increase of such strains may threaten the value of the nalidixic acid disk test to screen for reduced fluoroquinolone susceptibility in salmonellas.
TEXT
The fluoroquinolone susceptibility of Salmonella enterica isolates has been surveyed in the National Public Health Institute, Turku and Helsinki, Finland, since 1995 by analyzing 100 to 200 isolates yearly collected from Finnish travelers returning from abroad. Between 1995 and 1999, the annual proportion of reduced fluoroquinolone susceptibility increased significantly among all foreign isolates (6, 7). The increase was especially striking among isolates from travelers returning from Southeast Asia (6). From 1995 to 2002, all Salmonella isolates studied which showed reduced ciprofloxacin susceptibility were uniformly resistant to nalidixic acid. Based on these findings, we have validated the use of the nalidixic acid screening test in the detection of decreased fluoroquinolone susceptibility in salmonellas (5). Among 1,010 Salmonella isolates included in that study, identification of nalidixic acid resistance by the disk diffusion method provided a sensitivity of 100% and a specificity of 87.3% to screen for isolates with reduced susceptibility (5). In 2004, the use of the nalidixic acid screening test was recommended by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards) for the detection of invasive Salmonella isolates (14).
During our annual survey of fluoroquinolone susceptibility of Salmonella isolates in 2003, we focused attention on the emergence of a new quinolone resistance pattern among salmonellas originating from Southeast Asia. These isolates showed reduced ciprofloxacin susceptibility but were susceptible to nalidixic acid.
Susceptibility testing. The MICs of the isolates to ciprofloxacin and to nalidixic acid were determined by an Etest according to the manufacturer's instructions. In addition, the susceptibilities to these antimicrobials were determined by the disk diffusion test (13). Mueller-Hinton II agar (Becton Dickinson and Company, Sparks, MD) was used as the culture medium. Etests were purchased from AB Biodisk, Solna, Sweden, and the antimicrobial disks were from Oxoid Ltd. (Oxoid AB, Sollentuna, Sweden). The MIC breakpoints used for susceptibility and resistance to ciprofloxacin and nalidixic acid were 1 and 4 μg/ml and 8 and 32 μg/ml, respectively, as recommended by the Clinical and Laboratory Standards Institute (3). The corresponding inhibition zone diameters of the disk diffusion test for nalidixic acid were 19 and 13 mm (3). The MIC breakpoint value used for reduced ciprofloxacin susceptibility was 0.125 μg/ml. All measurements were repeated three times.
Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853 were used as controls in susceptibility testing. Also, three local Salmonella enterica strains were used as controls, one fully ciprofloxacin susceptible, one with reduced ciprofloxacin susceptibility, and one highly ciprofloxacin resistant.
Serotyping of the isolates was performed in the Enteric Bacteria Laboratory of the National Public Health Institute, Helsinki, Finland.
Pyrosequencing was applied to detect point mutations at the positions Ser83 and Asp87 in the quinolone resistance-determining region (QRDR) of the gyrA gene in salmonellas (20). Pyrosequencing was performed by using a PSQ 96 sample preparation kit, streptavidin-coated Sepharose beads (Amersham Biosciences, Piscataway, N.J.), a vacuum prep workstation, and a PSQ 96MA instrument (Biotage AB, Uppsala, Sweden) according to the instructions of the manufacturer. In one reaction, 20 μl of the PCR product and 15 pmol of a suitable sequencing primer were used (Table 1). The full details of the technique are available at http://www.pyrosequencing.com.
Characteristics of Salmonella isolates. During 2003 and 2004, we identified a total of 16 isolates showing a novel resistance pattern (Fig. 1 and Table 2). All of these isolates showed reduced ciprofloxacin susceptibility, with the MICs ranging between 0.125 and 0.5 μg/ml. The MICs of nalidixic acid varied from 4 to 16 μg/ml. Eight isolates were fully susceptible, and four isolates were intermediately resistant to nalidixic acid in all three measurements. For four isolates, the MICs varied between susceptible and intermediately resistant. The disk diffusion test results for nalidixic acid varied between susceptible and intermediately resistant. For two isolates, the inhibition zone diameter was 13 mm in one of three measurements.
All isolates were recovered from stool. Twelve isolates were from travelers returning from Thailand, and four isolates were from travelers returning from Malaysia. The isolates represented five different serotypes: Corvallis (n = 7), Stanley (n = 6), Mbandaka (n = 1), Montevideo (n = 1), and Virginia (n = 1). None of the isolates had a mutation at codon Ser83 or Asp87 in the QRDR of the gyrA gene.
These 16 isolates comprised 16.8% of the total 95 Salmonella isolates and 31.4% of the 51 isolates with reduced ciprofloxacin susceptibility recovered during the study period from patients returning from Thailand and Malaysia.
We describe here the emergence of a novel quinolone resistance pattern among Salmonella isolates originating from Southeast Asia. A population with such a resistance pattern has not been previously described. Anecdotal reports have been published on other enterobacteria showing similar resistance characteristics, but such strains have been solitary, as well as rare (12).
The existence and plausible future increase of Salmonella strains that are susceptible to nalidixic acid but show reduced susceptibility to ciprofloxacin may impede the detection of quinolone resistance of salmonellas in areas where such strains are common, since the nalidixic acid disk diffusion test may no longer be valid as a tool to screen for reduced fluoroquinolone susceptibility.
The mechanism of resistance in the Salmonella isolates described here is still unknown. The presence of five different serotypes among these non-fluoroquinolone-susceptible isolates excludes the possibility of the spread of only one clone. The absence of mutations at codon Ser83 or Asp87 of the QRDR of gyrA, which are the two most common sites of mutation leading to quinolone resistance (1, 4, 5, 6, 17, 19), suggests the involvement of other resistance mechanisms. The isolates were not characterized for other mutations; thus, mutations at other locations of the QRDR remain possible. Another plausible explanation for this type of resistance involves the presence of efflux pump mechanisms. It has been shown that exposure to low concentrations of fluoroquinolones may lead to activation of the efflux pump system and a reduction in susceptibility, even when there are no mutations in gyrA (2, 18). Moreover, a possible role of plasmid-mediated resistance (9) in these isolates must be kept in mind, considering that this resistance pattern was observed in multiple serovars.
We believe that the detection of low-level ciprofloxacin resistance in salmonellas is important for epidemiological, as well as for clinical, purposes. Although nontyphoidal Salmonella strains characteristically cause gastroenteritis, they can sometimes produce an invasive infection leading to serious complications. If such an infection is caused by a non-fluoroquinolone-susceptible Salmonella strain, treatment with another antimicrobial group may be indicated. Based on several reports, the use of fluoroquinolones in infections caused by Salmonella isolates with reduced susceptibility may lead to treatment failures (1, 8, 10, 11, 15-17, 21).
The Salmonella population described here may prove problematic, especially in those clinical laboratories that use the nalidixic acid disk diffusion test to screen for reduced ciprofloxacin susceptibility in salmonellas. It is recommended that at least invasive Salmonella isolates from travelers returning from Southeast Asia be tested by a fluoroquinolone Etest or another suitable MIC method.
Nucleotide sequence accession numbers. The nucleotide sequences determined in this study have been deposited in the EMBL database under accession numbers AM076385 to AM076400.
REFERENCES
Brown, J. C., C. J. Thomson, and S. G. Amyes. 1996. Mutations of the gyrA gene of clinical isolates of Salmonella typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quinolone drugs. J. Antimicrob. Chemother. 37:351-356.
Cebrián, L., J. C. Rodríguez, I. Escribano, and G. Royo. 2005. Characterization of Salmonella spp. mutants with reduced fluoroquinolone susceptibility: importance of efflux pump mechanisms. Chemotherapy 51:40-43.
Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing. Fifteenth informational supplement M100-S15. Clinical and Laboratory Standards Institute, Wayne, Pa.
Griggs, D. J., K. Gensberg, and L. J. Piddock. 1996. Mutations in gyrA gene of quinolone-resistant Salmonella serotypes isolated from humans and animals. Antimicrob. Agents Chemother. 40:1009-1013.
Hakanen, A., P. Kotilainen, J. Jalava, A. Siitonen, and P. Huovinen. 1999. Detection of decreased fluoroquinolone susceptibility in salmonellas and validation of nalidixic acid screening test. J. Clin. Microbiol. 37:3572-3577.
Hakanen, A., P. Kotilainen, P. Huovinen, H. Helenius, and A. Siitonen. 2001. Reduced fluoroquinolone susceptibility in Salmonella enterica serotypes in travelers returning from Southeast Asia. Emerg. Infect. Dis. 7:996-1003.
Hakanen, A., A. Siitonen, P. Kotilainen, and P. Huovinen. 1999. Increasing fluoroquinolone resistance in salmonella serotypes in Finland during 1995-1997. J. Antimicrob. Chemother. 43:145-148.
Helms, M., P. Vastrup, P. Gerner-Smidt, and K. Mlbak. 2002. Excess mortality associated with antimicrobial drug-resistant Salmonella typhimurium. Emerg. Infect. Dis. 8:490-495.
Jacoby, G. A. 2005. Mechanisms of resistance to quinolones. Clin. Infect. Dis. 41:120S-126S.
McCarron, B., and W. C. Love. 1997. Acalculous nontyphoidal salmonellal cholecystitis requiring surgical intervention despite ciprofloxacin therapy: report of three cases. Clin. Infect. Dis. 24:707-709.
Mlbak, K., D. L. Baggesen, F. M. Aarestrup, J. M. Ebbesen, J. Engberg, K. Frydendahl, P. Gerner-Smidt, A. M. Petersen, and H. C. Wegener. 1999. An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype Typhimurium DT104. N. Engl. J. Med. 341:1420-1425.
Moniot-Ville, N., J. Guibert, N. Moreau, J. F. Acar, E. Collatz, and L. Gutmann. 1991. Mechanisms of quinolone resistance in a clinical isolate of Escherichia coli highly resistant to fluoroquinolones but susceptible to nalidixic acid. Antimicrob. Agents Chemother. 35:519-523.
National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial disk susceptibility tests, 8th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement M100-S14. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Ouabdesselam, S., J. Tankovic, and C. J. Soussy. 1996. Quinolone resistance mutations in the gyrA gene of clinical isolates of Salmonella. Microb. Drug. Resist. 2:299-302.
Pers, C., P. Sogaard, and L. Pallesen. 1996. Selection of multiple resistance in Salmonella enteritidis during treatment with ciprofloxacin. Scand. J. Infect. Dis. 28:529-531.
Piddock, L. J., D. J. Griggs, M. C. Hall, and Y. F. Jin. 1993. Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients. Antimicrob. Agents Chemother. 37:662-666.
Piddock, L. J., Y. F. Jin, M. A. Webber, and M. J. Everett. 2002. Novel ciprofloxacin-resistant, nalidixic acid-susceptible mutant of Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2276-2278.
Reyna, F., M. Huesca, V. Gonzalez, and L. Y. Fuchs. 1995. Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance. Antimicrob. Agents Chemother. 39:1621-1623.
Ronaghi, M., M. Uhlen, and P. Nyren. 1998. A sequencing method based on real-time pyrophosphate. Science 281:363, 365.
Vasallo, F. J., P. Martin-Rabadan, L. Alcala, J. M. Garcia-Lechuz, M. Rodriguez-Creixems, and E. Bouza. 1998. Failure of ciprofloxacin therapy for invasive nontyphoidal salmonellosis. Clin. Infect. Dis. 26:535-536.(Antti J. Hakanen, Mariann)