- Email: [email protected]
Decrease in the incidence of mupirocin resistance among methicillin-resistant Staphylococcus aureus in carriers from an intensive care unit
Decrease in the incidence of mupirocin resistance among methicillin-resistant Staphylococcus aureus in carriers from an intensive care unit Juliana Caiera˜o, MSc,a Laura Berquo´, RC, MSc,b Cı´cero Dias, MSc,a,b and Pedro Alves d’Azevedo, PhDa Porto Alegre, RS, Brazil
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a serious nosocomial problem, globally distributed. Decolonization with mupirocin can be used to control its dissemination. Objective: To determine the incidence of mupirocin resistance among MRSA carriers from an intensive care unit. Methods: We obtained 2723 nasal swabs during 3 years. Resistance to methicillin and mupirocin were verified (agar diffusion and the E test) and confirmed by polymerase chain reaction (PCR) (mecA for methicillin; ileS-2 and mupA for mupirocin). Plasmidcuring procedure and pulsed-field gel electrophoresis (PFGE) were employed in isolates exhibiting high resistance to mupirocin (HR-Mup) and in other selected organisms. Results: The overall incidence of HR-Mup among MRSA carriers during the studied period was 4.84% (8/165); however, the incidence decreased from 13.04% (6/46) in the first year to 3.5% (2/57) in the second year and was 0% in the last year (P 5 .02). LR-Mup, in contrast, increased significantly (P 5 .01). Conclusion: Plasmid-curing procedure showed the plasmid location of genes responsible for HR-Mup. PFGE demonstrated that most MRSA, including the isolates with HR-Mup, were genetically related. The decline in HR-Mup may be attributable to the plasmid location of genes (ileS-2/mupA) and to the fact that all patients colonized with HR-Mup MRSA died or were discharged in a relatively short period of time. (Am J Infect Control 2006;34:6-9.)
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a serious problem in hospitals and are globally distributed.1 Infection control programs implement control measures to contain the dissemination of resistant strains and include efforts to eradicate carriage of S aureus.2 Carriage may be eradicated by topical application of mupirocin on the anterior nares of individuals recognized as MRSA carriers. Some infection control programs use decolonization as a component of the process of MRSA control.3 Mupirocin inhibits bacterial isoleucyl-t-RNA synthetase and, as a consequence, protein synthesis.4,5 Resistance to mupirocin among staphylococci, however, is an emerging worldwide problem and may contribute to failures in the eradication of MRSA.6,7 In Brazil, the presence of MRSA with resistance to mupirocin was documented in the last decade.8
From the Fundac xa˜o Faculdade Federal de Cieˆncias Me´dicas de Porto Alegrea and Hospital Ma˜e de Deus,b Porto Alegre, Brazil. Reprint requests: Cı´cero Dias, MD, Fundac xa˜o Faculdade Federal de Cieˆncias Me´dicas de Porto Alegre, Rua Sarmento Leite, 245 Centro, 90050170 Porto Alegre, RS, Brazil. E-mail: [email protected]. 0196-6553/$32.00 Copyright ª 2006 by the Association for Professionals in Infection Control and Epidemiology, Inc. doi:10.1016/j.ajic.2005.08.006
6
Two levels of resistance are recognized: strains exhibiting minimal inhibitory concentrations (MIC) $8 to 128 mg/mL are considered of low-level resistance to mupirocin (LR-Mup) and are not clinically relevant. On the other hand, strains with MIC $256 mg/mL are resistant to high levels of mupirocin (HR-Mup). These strains produce an additional isoleucyl-t-RNA synthetase that confers resistance to mupirocin.9 The genetic determinant of this type of resistance is typically located in plasmids10 but is occasionally located in the chromosomal DNA.11 The presence of this determinants may be identified by polymerase chain reaction (PCR) using 2 pairs of primers for ileS-212 or mupA.11 The objective of this study was to determine the incidence of nasal carriage of mupirocin resistance among MRSA from patients of an adult intensive care unit (ICU) and to investigate genetic determinants in strains presenting resistance to this agent.
METHODS Hospital Ma˜e de Deus, Porto Alegre, Brazil, is a general, 200-bed hospital with 2 adult ICUs (21 beds). A surveillance program to detect MRSA carriers among adult ICU patients has been active since 1996. All carriers were submitted to a protocol that included contact precautions,13 daily chlorexidine baths (2% soap), and treatment with topical mupirocin applied on the anterior nares (3 times/day, during 5 days). Mupirocin is
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Table 1. New cases of MRSA from patients in adult ICUs and resistance to mupirocin in the Hospital Ma˜e de Deus, Porto Alegre, Brazil MRSA Period 2000-2001 2001-2002 2002-2003 Total
HR-Mup in MRSA y
LR-Mup in MRSA
No. of swabs obtained
n
Incidence* (%)
n
Incidence (%)
n
Incidencez (%)
905 894 924 2723
46 57 62 165
5.08 6.37 6.7 6.06
6 2 0 8
13.04 (6/46) 3.5 (2/57) 0 (0/62) 4.84 (8/165)
3 5 14 22
6.52 (3/46) 8.77 (5/57) 22.5 (14/62) 13.3 (22/165)
MRSA, Methicillin-resistant Staphylococcus aureus; HR-Mup, resistance to high levels of mupirocin (MIC $256 mg/mL); LR-Mup: resistance to low levels of mupirocin (MIC $ 8-128 mg/mL). *P 5 .3, x2 for trend. y P 5 .02, x2 for trend. z P 5 .01, x2 for trend.
used exclusively in ICU MRSA carriers in our institution. In the present study, all adult patients in ICUs were surveyed from May 2000 to April 2003. Swabs (CB Products, Sa˜o Paulo, Brazil) were obtained weekly from the anterior nares of individuals and plated on trypticase soy agar with 5% sheep blood (BioMe´rieux, Rio de Janeiro, Brazil) and on oxacillin-resistant screening agar, supplemented with oxacillin (2.0 mg/mL) and polymyxin B (50,000 IU/L; Oxoid Limited, Basingstoke, United Kingdom). To determine incidence of mupirocin resistance among MRSA, isolates from all newly identified carriers were evaluated. A disk diffusion procedure was used to define resistance to methicillin, following recommendations from the National Committee for Clinical Laboratory Standards.14 Isolates from newly identified carriers were submitted to the E test to obtain MICs to mupirocin, using previously recommended interpretive criteria.15 The presence of genes associated with mupirocin resistance was investigated by PCR in all MRSA isolates that showed an MIC $8 mg/mL to mupirocin and in 10 MRSA randomly selected susceptible isolates (MIC ,8 mg/mL). The following primers were used: ileS-2 1: 5’-GTT TAT CTT CTG ATG CTG AG, ileS-2 2: 5’-CCC CAG TTA CAC CGA TAT AA11; mupA 1: 5’-TGA CAA TAG AAA AGG ACA GG, mupA 2: 5’-CTC TAA TTC AAC TGG TAA GCC.15 To confirm resistance to methicillin, a PCR procedure was performed to detect the presence of the mecA gene, using the primers mecA 1: 5’-TGG CTA TCG TGT CAC AAT CG and mecA 2: 5’-CTG GAA CTT GTT GAG CAG AG.16 Primers amplified segments of 237, 190, and 310 bp, of the genes ileS-2, mupA, and mecA, respectively, that were visualized after electrophoresis on 2% agarose containing ethidium bromide. Isolates showing HR-Mup or the presence of one of the resistance genes detected by PCR were submitted to a plasmid curing procedure.17 From day 5 to day 12, isolates were passaged daily (tryptic soy broth, 42C), and susceptibility testing was done on days 6 through 12 to determine whether the mupirocin MIC changed.
The presence of the genes ileS-2 and mupA was verified by PCR. A selected group of organisms, including 7 of 8 isolates with HR-Mup, representatives of LR-Mup, and isolates susceptible to mupirocin, was subjected to macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). Additionally, a strain representative of the major MRSA clone in Brazil (SpA1721) was included for comparative purposes. Enzyme SmaI was used in the restriction, and electrophoresis was performed in a CHEF-DRII apparatus (Bio-Rad, Richmond, CA). The following conditions were employed: 0.5 Tris-borate-EDTA, 1% agarose, 13C, and 200 V. The electrophoresis was run for 23 hours, and the switch interval was ramped from 5 to 60 seconds. Gels were stained with ethidium bromide and visually inspected. Isolates were considered related if there were 4 to 6 fragment differences in the profiles and as closely related (subtypes) if there were 2 or 3 differences observed.18 Charts of patients carrying HR-Mup were electronically reviewed. A x2 test for trend was used to compare differences in the incidence of HR-Mup according to the period studied.
RESULTS The distribution of results during the 3-year period of study appears in Table 1. Between May 2000 and April 2003, 2723 swabs were collected from ICU patients. Among these patients, the incidence of MRSA was 6.6% (165/2723). This incidence was variable as follows: 5.08% (46/905), 6.37% (57/894), and 6.7% (62/924) in the first, second, and third year of study, respectively. Eight MRSA isolates had mupirocin MICs $256 mg/mL (HR-Mup), whereas 22 exhibited LRMup (MICs ranging from 8.0 to 32.0 mg/mL). The global incidence of HR-Mup among MRSA was 4.84% (8/165), and, during the 3-year period, it decreased from 13.04% (6/46) in the first year to 3.5% (2/57) in the second year and to 0% in the third year (P 5 .02). In contrast, LR-Mup increased significantly (P 5 .01).
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Resistance to methicillin was confirmed by detecting the mecA gene in all MRSA detected by the phenotypic method. The presence of ileS-2 was observed in all isolates with HR-Mup and in 1 isolate with LRMup (MIC 5 32 mg/mL). PCR for mupA was positive in 7 of 8 HR-Mup isolates and in none of the isolates exhibiting LR-Mup. Discrepant results were retested and confirmed. All 10 randomly selected susceptible isolates were negative for the presence of both the ileS-2 and the mupA gene. After the plasmid-curing procedure, all 8 isolates, which initially had the HR-Mup phenotype, had MIC ,8.0 mg/mL, and the ileS-2 and mupA were not detected by PCR. The isolate that initially had a MIC 5 32 mg/mL and carried the ileS-2 but not the mupA by PCR also became susceptible to mipirocin after plasmid curing, and the ileS-2 was no longer detected. Three distinctive PFGE patterns, A, B, and C, were observed. Pattern A was observed in 13 isolates (5 with HR-Mup, 5 with LR-Mup, in 2 susceptible isolates, and in the strain Sp-A1721, representative of the Brazilian clone). Patterns B and C, possibly related to pattern A, were observed in isolates with HR-Mup (1 isolate each) (image not shown). One isolate with HR-Mup was not available for genotyping.
DISCUSSION Mupirocin resistance is disseminated among Staphylococcus aureus, being found among methicillin-resistant and susceptible isolates.6-8 The actual, or at least potential, development of mupirocin resistance is a drawback for programs that use this agent to eradicate MRSA carriage.19 In this study we followed the incidence of mupirocin resistance during a 3-year period among MRSA carriers in an intensive care unit. Although disk diffusion with methicillin is not the reference method to define MRSA, our primary isolation was done on selective medium, improving the sensitivity of MRSA detection. In the first year of the study (May 2000-April 2001), among 46 MRSA isolates, 6 presented HR-Mup, corresponding to an incidence of 13.04% (6 MRSA with HR-Mup among 46 MRSA isolated during this year). Only 2 isolates showed HR-Mup, decreasing the incidence to 3.5% (2/57) in the second year of the study. In the remaining year, HR-Mup was not detected among 62 MRSA isolates (P 5 .02). Infectious disease specialists, microbiologists, and epidemiologists are familiar with an opposite situation: the emergence of bacterial resistance to antimicrobial agents. Some studies, however, show that mupirocin resistance can remain stable or even decrease. Over a 4-year period, the prevalence of HR-Mup was kept below 2% among 1200 S aureus isolates from patients in Greek
Caiera˜o et al
hospitals.20 At another hospital, the incidence of mupirocin resistance (both HR-Mup and LR-Mup) in MRSA declined after the facility implemented administrative controls for mupirocin prescriptions.21 It is important to mention that, in our institution, the use of mupirocin is directly controlled by the infection control service, and no modification in the policy of prescription was done during the period of the study. Most MRSA isolates belong to a few clones that have been disseminated over wide geographic areas. In fact, all isolates that we submitted to genotyping were at least possibly related, independent of resistance to mupirocin. Also, most of the isolates tested were closely related to the representative of the Brazilian clone. All isolates with the phenotype HR-Mup lost the resistance after the plasmid-curing experiment. Clearly, HR-Mup isolates of this study had the genetic determinant of resistance located in plasmids. This may be partially associated with the decrease in resistance because plasmidborne resistance is more labile, and the dominant MRSA clone could persist without mupirocin resistance. Five of 8 (62.5%) patients with HR-Mup died during the year after their isolates were identified. None of the 3 surviving patients were readmitted to the hospital after April 2002. It should be emphasized that HR-Mup may be found in S aureus susceptible to methicillin, and we did not search for mupirocin resistance among these organisms. LR-Mup, on the other hand, increased significantly during the 3 years of the study (P 5 .01). This phenotype is attributed to chromosomal mutations that do not produce HR-Mup. It is important to consider that, with a single exception, all isolates with LO-Mup did not present positive results for the mupA and ileS-2 by PCR. The clinical relevance of this finding and its implications in control of MRSA remain to be elucidated. The isolate presenting the discrepant behavior mentioned above had MIC 5 32 mg/mL to mupirocin and was positive for ileS-2. After being submitted to plasmid curing, the isolate presented the susceptible phenotype, and the ileS-2 gene was no longer present. Also, an isolate with HR-Mup showed a negative PCR reaction when tested for the mupA gene. Because the primers used in the study amplified different sequences of the same mupirocin-resistant gene, it is possible that this isolate presents minor DNA sequence variation in the region amplified by mupA. The results of these isolates may be further investigated in detail. In summary, in this surveillance study, we report an unusual finding: the decrease in the incidence of HRMup in MRSA obtained from carriers of an intensive care unit, during a period of regular use of mupirocin. We can conclude that the dynamics of mupirocin resistance may be particular of each institution. Two elements must be considered in the decline in
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mupirocin resistance in this study: the plasmid location of genes responsible for resistance and the relatively short period of permanence of patients carrying HR-Mup MRSA in the institution. The authors thank Dr. Suzane Silbert, from the Universidade Federal de Sa˜o Paulo, for expertise in pulsed-field gel electrophoresis.
References 1. Diekema DJ, Pfaller MA, Turnidge J, Verhoef J, Bell J, Fluit AC, et al, and the Sentry Participants Group. Genetic relatedness of multidrug-resistant, methicillin (oxacillin)-resistant Staphylococcus aureus bloodstream isolates from SENTRY antimicrobial resistance centers worldwide, 1998. Microb Drug Resist 2000;6:213-21. 2. Girou E, Pujade G, Legrand P, Cizeau F, Brun-Buisson C. Selective screening of carriers for control of methicillin-resistant Staphylococcus aureus (MRSA) in high-risk hospital areas with a high level of endemic MRSA. Clin Infect Dis 1998;27:543-50. 3. Perl T, Cullen JJ, Wenzel RP, Zimmerman MB, Pfaller MA, Sheppard D, et al, and the Mupirocin and the Risk of Staphylococcus aureus Study Team. Intranasal mupirocin to prevent postoperative Staphylococcus aureus infections. N Engl J Med 2002;346:1871-7. 4. Hughes J, Mellows G. Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid. J Biochem 1978; 176:305-18. 5. Morton TM, Johnston JL, Patterson J, Archer GL. Characterization of a conjugative staphylococcal mupirocin-resistant plasmid. Antimicrob Agents Chemother 1995;39:1272-80. 6. Upton A, Lang S, Heffernan H. Mupirocin and Staphylococcus aureus: a recent paradigm of emerging antibiotic resistance. J Antimicrob Chemother 2003;51:613-7. 7. Chaves F, Garcı´a-Martı´nez J, de Miguel S, Otero J. Molecular characterization of resistance to mupirocin in methicillin-susceptible and -resistant isolates of Staphylococcus aureus from nasal samples. J Clin Microbiol 2004;42:822-4. 8. dos Santos KR, Teixeira LM, Bravo Neto GP, Fonseca LS, Gontijo Filho PP. Mupirocin- and methicillin-resistant Staphylococcus aureus spreading in an intermediate-care unit in a Brazilian hospital. Infect Control Hosp Epidemiol 1998;19:622-3. 9. Gilbart J, Perry CR, Slocombe B. High-level mupirocin resistance in Staphylococcus aureus: evidence for two distinct isoleucyl-tRNA synthetases. Antimicrob Agents Chemother 1993;37:32-8.
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10. Udo EE, Jacob LE, Mathew B. Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance. J Med Microbiol 2001;50:909-15. 11. Udo EE, Al-Sweih N, Noronha BC. A chromosomal location of the mupA gene in Staphylococcus aureus expressing high-level mupirocin resistance. J Antimicrob Chemother 2003;51:1283-6. 12. Nunes EL, dos Santos KR, Mondino PJ, Bastos MC, Giambiagi de Marval M. Detection of the ileS-2 gene encoding mupirocin resistance in methicillin-resistant Staphylococcus aureus by multiplex PCR. Diagn Microbiol Infect Dis 1999;34:77-81. 13. The Hospital Infection Control Practices Advisory Committee, Center for Disease Control and Prevention. Public Health Service, US Department of Health and Human Services. Guideline for isolation precautions in hospitals. Part II: Recommendations for isolation precautions in hospitals. Am J Infect Control 1996;24:32-52. 14. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing; twelfth informational supplement M100-S12. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. 15. Palepou MF, Johnson AP, Cookson BD, Beattie H, Charlett A, Woodford N. Evaluation of disk diffusion and E test for determining the susceptibility of Staphylococcus aureus to mupirocin. J Antimicrob Chemother 1998;42:577-83. 16. Vannuffel P, Laterre PF, Bouyer M, Gigi J, Vandercam B, Reynaert M, et al. Rapid and specific molecular identification of methicillin-resistant Staphylococcus aureus in endotracheal aspirates from mechanically ventilated patients. J Clin Microbiol 1998;36:2366-8. 17. Walker ES, Vasquez JE, Dula R, Bullock H, Sarubbi FA. Mupirocinresistant, methicillin-resistant Staphylococcus aureus: does mupirocin remain effective? Infect Control Hosp Epidemiol 2003;24:342-6. 18. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9. 19. Vasquez JE, Walker ES, Franzus BW, Overbay BK, Reagan DR, Sarubbi FA. The epidemiology of mupirocin resistance among methicillin-resistant Staphylococcus aureus at a Veterans’ Affairs hospital. Infect Control Hosp Epidemiol 2000;21:459-64. 20. Petinaki E, Spiliopoulou I, Kontos F, Maniati M, Bersos Z, Stakias N, et al. Clonal dissemination of mupirocin-resistant staphylococci in Greek hospitals. J Antimicrob Chemother 2004;53:105-8. 21. Walker ES, Levy F, Shorman M, David G, Abdala J, Sarubbi FA. A decline in mupirocin resistance in methicillin-resistant Staphylococcus aureus accompanied administrative control of prescriptions. J Clin Microbiol 2004;42:2792-5.
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a serious nosocomial problem, globally distributed. Decolonization with mupirocin can be used to control its dissemination. Objective: To determine the incidence of mupirocin resistance among MRSA carriers from an intensive care unit. Methods: We obtained 2723 nasal swabs during 3 years. Resistance to methicillin and mupirocin were verified (agar diffusion and the E test) and confirmed by polymerase chain reaction (PCR) (mecA for methicillin; ileS-2 and mupA for mupirocin). Plasmidcuring procedure and pulsed-field gel electrophoresis (PFGE) were employed in isolates exhibiting high resistance to mupirocin (HR-Mup) and in other selected organisms. Results: The overall incidence of HR-Mup among MRSA carriers during the studied period was 4.84% (8/165); however, the incidence decreased from 13.04% (6/46) in the first year to 3.5% (2/57) in the second year and was 0% in the last year (P 5 .02). LR-Mup, in contrast, increased significantly (P 5 .01). Conclusion: Plasmid-curing procedure showed the plasmid location of genes responsible for HR-Mup. PFGE demonstrated that most MRSA, including the isolates with HR-Mup, were genetically related. The decline in HR-Mup may be attributable to the plasmid location of genes (ileS-2/mupA) and to the fact that all patients colonized with HR-Mup MRSA died or were discharged in a relatively short period of time. (Am J Infect Control 2006;34:6-9.)
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a serious problem in hospitals and are globally distributed.1 Infection control programs implement control measures to contain the dissemination of resistant strains and include efforts to eradicate carriage of S aureus.2 Carriage may be eradicated by topical application of mupirocin on the anterior nares of individuals recognized as MRSA carriers. Some infection control programs use decolonization as a component of the process of MRSA control.3 Mupirocin inhibits bacterial isoleucyl-t-RNA synthetase and, as a consequence, protein synthesis.4,5 Resistance to mupirocin among staphylococci, however, is an emerging worldwide problem and may contribute to failures in the eradication of MRSA.6,7 In Brazil, the presence of MRSA with resistance to mupirocin was documented in the last decade.8
From the Fundac xa˜o Faculdade Federal de Cieˆncias Me´dicas de Porto Alegrea and Hospital Ma˜e de Deus,b Porto Alegre, Brazil. Reprint requests: Cı´cero Dias, MD, Fundac xa˜o Faculdade Federal de Cieˆncias Me´dicas de Porto Alegre, Rua Sarmento Leite, 245 Centro, 90050170 Porto Alegre, RS, Brazil. E-mail: [email protected]. 0196-6553/$32.00 Copyright ª 2006 by the Association for Professionals in Infection Control and Epidemiology, Inc. doi:10.1016/j.ajic.2005.08.006
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Two levels of resistance are recognized: strains exhibiting minimal inhibitory concentrations (MIC) $8 to 128 mg/mL are considered of low-level resistance to mupirocin (LR-Mup) and are not clinically relevant. On the other hand, strains with MIC $256 mg/mL are resistant to high levels of mupirocin (HR-Mup). These strains produce an additional isoleucyl-t-RNA synthetase that confers resistance to mupirocin.9 The genetic determinant of this type of resistance is typically located in plasmids10 but is occasionally located in the chromosomal DNA.11 The presence of this determinants may be identified by polymerase chain reaction (PCR) using 2 pairs of primers for ileS-212 or mupA.11 The objective of this study was to determine the incidence of nasal carriage of mupirocin resistance among MRSA from patients of an adult intensive care unit (ICU) and to investigate genetic determinants in strains presenting resistance to this agent.
METHODS Hospital Ma˜e de Deus, Porto Alegre, Brazil, is a general, 200-bed hospital with 2 adult ICUs (21 beds). A surveillance program to detect MRSA carriers among adult ICU patients has been active since 1996. All carriers were submitted to a protocol that included contact precautions,13 daily chlorexidine baths (2% soap), and treatment with topical mupirocin applied on the anterior nares (3 times/day, during 5 days). Mupirocin is
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Table 1. New cases of MRSA from patients in adult ICUs and resistance to mupirocin in the Hospital Ma˜e de Deus, Porto Alegre, Brazil MRSA Period 2000-2001 2001-2002 2002-2003 Total
HR-Mup in MRSA y
LR-Mup in MRSA
No. of swabs obtained
n
Incidence* (%)
n
Incidence (%)
n
Incidencez (%)
905 894 924 2723
46 57 62 165
5.08 6.37 6.7 6.06
6 2 0 8
13.04 (6/46) 3.5 (2/57) 0 (0/62) 4.84 (8/165)
3 5 14 22
6.52 (3/46) 8.77 (5/57) 22.5 (14/62) 13.3 (22/165)
MRSA, Methicillin-resistant Staphylococcus aureus; HR-Mup, resistance to high levels of mupirocin (MIC $256 mg/mL); LR-Mup: resistance to low levels of mupirocin (MIC $ 8-128 mg/mL). *P 5 .3, x2 for trend. y P 5 .02, x2 for trend. z P 5 .01, x2 for trend.
used exclusively in ICU MRSA carriers in our institution. In the present study, all adult patients in ICUs were surveyed from May 2000 to April 2003. Swabs (CB Products, Sa˜o Paulo, Brazil) were obtained weekly from the anterior nares of individuals and plated on trypticase soy agar with 5% sheep blood (BioMe´rieux, Rio de Janeiro, Brazil) and on oxacillin-resistant screening agar, supplemented with oxacillin (2.0 mg/mL) and polymyxin B (50,000 IU/L; Oxoid Limited, Basingstoke, United Kingdom). To determine incidence of mupirocin resistance among MRSA, isolates from all newly identified carriers were evaluated. A disk diffusion procedure was used to define resistance to methicillin, following recommendations from the National Committee for Clinical Laboratory Standards.14 Isolates from newly identified carriers were submitted to the E test to obtain MICs to mupirocin, using previously recommended interpretive criteria.15 The presence of genes associated with mupirocin resistance was investigated by PCR in all MRSA isolates that showed an MIC $8 mg/mL to mupirocin and in 10 MRSA randomly selected susceptible isolates (MIC ,8 mg/mL). The following primers were used: ileS-2 1: 5’-GTT TAT CTT CTG ATG CTG AG, ileS-2 2: 5’-CCC CAG TTA CAC CGA TAT AA11; mupA 1: 5’-TGA CAA TAG AAA AGG ACA GG, mupA 2: 5’-CTC TAA TTC AAC TGG TAA GCC.15 To confirm resistance to methicillin, a PCR procedure was performed to detect the presence of the mecA gene, using the primers mecA 1: 5’-TGG CTA TCG TGT CAC AAT CG and mecA 2: 5’-CTG GAA CTT GTT GAG CAG AG.16 Primers amplified segments of 237, 190, and 310 bp, of the genes ileS-2, mupA, and mecA, respectively, that were visualized after electrophoresis on 2% agarose containing ethidium bromide. Isolates showing HR-Mup or the presence of one of the resistance genes detected by PCR were submitted to a plasmid curing procedure.17 From day 5 to day 12, isolates were passaged daily (tryptic soy broth, 42C), and susceptibility testing was done on days 6 through 12 to determine whether the mupirocin MIC changed.
The presence of the genes ileS-2 and mupA was verified by PCR. A selected group of organisms, including 7 of 8 isolates with HR-Mup, representatives of LR-Mup, and isolates susceptible to mupirocin, was subjected to macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). Additionally, a strain representative of the major MRSA clone in Brazil (SpA1721) was included for comparative purposes. Enzyme SmaI was used in the restriction, and electrophoresis was performed in a CHEF-DRII apparatus (Bio-Rad, Richmond, CA). The following conditions were employed: 0.5 Tris-borate-EDTA, 1% agarose, 13C, and 200 V. The electrophoresis was run for 23 hours, and the switch interval was ramped from 5 to 60 seconds. Gels were stained with ethidium bromide and visually inspected. Isolates were considered related if there were 4 to 6 fragment differences in the profiles and as closely related (subtypes) if there were 2 or 3 differences observed.18 Charts of patients carrying HR-Mup were electronically reviewed. A x2 test for trend was used to compare differences in the incidence of HR-Mup according to the period studied.
RESULTS The distribution of results during the 3-year period of study appears in Table 1. Between May 2000 and April 2003, 2723 swabs were collected from ICU patients. Among these patients, the incidence of MRSA was 6.6% (165/2723). This incidence was variable as follows: 5.08% (46/905), 6.37% (57/894), and 6.7% (62/924) in the first, second, and third year of study, respectively. Eight MRSA isolates had mupirocin MICs $256 mg/mL (HR-Mup), whereas 22 exhibited LRMup (MICs ranging from 8.0 to 32.0 mg/mL). The global incidence of HR-Mup among MRSA was 4.84% (8/165), and, during the 3-year period, it decreased from 13.04% (6/46) in the first year to 3.5% (2/57) in the second year and to 0% in the third year (P 5 .02). In contrast, LR-Mup increased significantly (P 5 .01).
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Resistance to methicillin was confirmed by detecting the mecA gene in all MRSA detected by the phenotypic method. The presence of ileS-2 was observed in all isolates with HR-Mup and in 1 isolate with LRMup (MIC 5 32 mg/mL). PCR for mupA was positive in 7 of 8 HR-Mup isolates and in none of the isolates exhibiting LR-Mup. Discrepant results were retested and confirmed. All 10 randomly selected susceptible isolates were negative for the presence of both the ileS-2 and the mupA gene. After the plasmid-curing procedure, all 8 isolates, which initially had the HR-Mup phenotype, had MIC ,8.0 mg/mL, and the ileS-2 and mupA were not detected by PCR. The isolate that initially had a MIC 5 32 mg/mL and carried the ileS-2 but not the mupA by PCR also became susceptible to mipirocin after plasmid curing, and the ileS-2 was no longer detected. Three distinctive PFGE patterns, A, B, and C, were observed. Pattern A was observed in 13 isolates (5 with HR-Mup, 5 with LR-Mup, in 2 susceptible isolates, and in the strain Sp-A1721, representative of the Brazilian clone). Patterns B and C, possibly related to pattern A, were observed in isolates with HR-Mup (1 isolate each) (image not shown). One isolate with HR-Mup was not available for genotyping.
DISCUSSION Mupirocin resistance is disseminated among Staphylococcus aureus, being found among methicillin-resistant and susceptible isolates.6-8 The actual, or at least potential, development of mupirocin resistance is a drawback for programs that use this agent to eradicate MRSA carriage.19 In this study we followed the incidence of mupirocin resistance during a 3-year period among MRSA carriers in an intensive care unit. Although disk diffusion with methicillin is not the reference method to define MRSA, our primary isolation was done on selective medium, improving the sensitivity of MRSA detection. In the first year of the study (May 2000-April 2001), among 46 MRSA isolates, 6 presented HR-Mup, corresponding to an incidence of 13.04% (6 MRSA with HR-Mup among 46 MRSA isolated during this year). Only 2 isolates showed HR-Mup, decreasing the incidence to 3.5% (2/57) in the second year of the study. In the remaining year, HR-Mup was not detected among 62 MRSA isolates (P 5 .02). Infectious disease specialists, microbiologists, and epidemiologists are familiar with an opposite situation: the emergence of bacterial resistance to antimicrobial agents. Some studies, however, show that mupirocin resistance can remain stable or even decrease. Over a 4-year period, the prevalence of HR-Mup was kept below 2% among 1200 S aureus isolates from patients in Greek
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hospitals.20 At another hospital, the incidence of mupirocin resistance (both HR-Mup and LR-Mup) in MRSA declined after the facility implemented administrative controls for mupirocin prescriptions.21 It is important to mention that, in our institution, the use of mupirocin is directly controlled by the infection control service, and no modification in the policy of prescription was done during the period of the study. Most MRSA isolates belong to a few clones that have been disseminated over wide geographic areas. In fact, all isolates that we submitted to genotyping were at least possibly related, independent of resistance to mupirocin. Also, most of the isolates tested were closely related to the representative of the Brazilian clone. All isolates with the phenotype HR-Mup lost the resistance after the plasmid-curing experiment. Clearly, HR-Mup isolates of this study had the genetic determinant of resistance located in plasmids. This may be partially associated with the decrease in resistance because plasmidborne resistance is more labile, and the dominant MRSA clone could persist without mupirocin resistance. Five of 8 (62.5%) patients with HR-Mup died during the year after their isolates were identified. None of the 3 surviving patients were readmitted to the hospital after April 2002. It should be emphasized that HR-Mup may be found in S aureus susceptible to methicillin, and we did not search for mupirocin resistance among these organisms. LR-Mup, on the other hand, increased significantly during the 3 years of the study (P 5 .01). This phenotype is attributed to chromosomal mutations that do not produce HR-Mup. It is important to consider that, with a single exception, all isolates with LO-Mup did not present positive results for the mupA and ileS-2 by PCR. The clinical relevance of this finding and its implications in control of MRSA remain to be elucidated. The isolate presenting the discrepant behavior mentioned above had MIC 5 32 mg/mL to mupirocin and was positive for ileS-2. After being submitted to plasmid curing, the isolate presented the susceptible phenotype, and the ileS-2 gene was no longer present. Also, an isolate with HR-Mup showed a negative PCR reaction when tested for the mupA gene. Because the primers used in the study amplified different sequences of the same mupirocin-resistant gene, it is possible that this isolate presents minor DNA sequence variation in the region amplified by mupA. The results of these isolates may be further investigated in detail. In summary, in this surveillance study, we report an unusual finding: the decrease in the incidence of HRMup in MRSA obtained from carriers of an intensive care unit, during a period of regular use of mupirocin. We can conclude that the dynamics of mupirocin resistance may be particular of each institution. Two elements must be considered in the decline in
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mupirocin resistance in this study: the plasmid location of genes responsible for resistance and the relatively short period of permanence of patients carrying HR-Mup MRSA in the institution. The authors thank Dr. Suzane Silbert, from the Universidade Federal de Sa˜o Paulo, for expertise in pulsed-field gel electrophoresis.
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