Vol. 28, No. 9
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p. 1898-1902
0095-1137/90/091898-05$02.00/0 Copyright © 1990, American Society for Microbiology
Staphylococcus aureus Colonization and Infection in Patients Continuous Ambulatory Peritoneal Dialysis
on
A. PIGNATARI,"12 M. PFALLER,l13* R. HOLLIS,' R. SESSO,4 I. LEME,2 AND L. HERWALDT5 Departments of Pathology' and Internal Medicine,5 University of lowa College of Medicine, Iowa City, Iowa 52242; Department of Veterans Affairs Medical Center, Iowa City, Iowa 522403; and Disciplina de Doencas Infecciosas e Parasitarias2 and Disciplina de Nefrologia,4 Escola Paulista de Medicina, Sao Paulo, Brazil Received 22 February 1990/Accepted 11 June 1990
Staphylococcus aureus is the most common cause of peritonitis in patients undergoing peritoneal dialysis in Brazil. Using restriction endonuclease analysis of plasmid DNA, we investigated the importance of chronic carriage of S. aureus in the development of peritonitis in patients on continuous ambulatory peritoneal dialysis at the Division of Nephrology, Escola Paulista de Medicina, Sao Paulo, Brazil. A total of 117 isolates (30 patients) of S. aureus were available for typing, including 51 isolates (22 patients) from the nares, 58 isolates (27 patients) from pericatheter skin, and 8 isolates (6 patients) from peritoneal fluid, from patients with peritonitis. Restriction endonuclease subtyping showed that although most patients harbored more than one subtype of S. aureus, in the majority of patients nasal and/or pericatheter skin isolates with identical restriction endonuclease digest patterns were recovered on more than one occasion. Furthermore, 95% of patients with both nasal and pericatheter colonization were colonized with the same subtypes at both sites. All of the patients with peritonitis were infected with a subtype which colonized the nares, pericatheter skin, or both. These results demonstrate the importance of an endogenous source of S. aureus in the development of continuous ambulatory peritoneal dialysis-associated peritonitis. MATERIALS AND METHODS
Peritonitis is a major complication in patients on continuous ambulatory peritoneal dialysis (CAPD) (3, 4, 9, 12-14). In many countries, the most common cause of peritonitis in patients on CAPD is coagulase-negative staphylococci (4, 6,
Patients. All 43 patients on CAPD at the Division of Nephrology, Escola Paulista de Medicina (Sao Paulo, Brazil), from April 1987 through June 1988 were included in the surveillance study. The clinical characteristics of these patients have been described previously (13). Surveillance samples from the nares and pericatheter skin were obtained every 2 months (sampling period). Samples of peritoneal dialysis fiuid were also cultured when it was clinically indicated for the diagnosis of peritonitis. Only patients with at least two samples that were positive for S. aureus were included in the present study. Definitions. Peritonitis was defined as cloudy dialysis effluent containing more than 100 leukocytes per mm3, with more than 50% of these cells being polymorphonuclear leukocytes. The presence of an organism on Gram staining or a positive culture was confirmatory but not necessary for the diagnosis of peritonitis. Microbiology. Surveillance samples of the nares and pericatheter skin were obtained by using sterile, premoistened calcium alginate swabs (Cefar-Farmaco Diagnostic Ltda., Sao Paulo, Brazil) and were transported to the microbiology laboratory, where they were immediately streaked onto plates containing tryptic soy agar with 5% sheep blood, MacConkey agar, and mannitol-salt agar (Difco Laboratories, Detroit, Mich.). If patients developed peritonitis, 5 ml of peritoneal fluid was inoculated into each of two bottles (Hemo-cult 1; Laborclin Ltda., Parana, Brazil) containing 50 ml of tryptic soy broth (Difco). All cultures were incubated at 36°C for 48 h and examined daily for evidence of growth. Gram-positive cocci which produced catalase and coagulase were identified as S. aureus. Isolates of S. aureus were saved on nutrient agar slants and sent to the Special Microbiology Laboratory at the University of Iowa for typing by restriction endonuclease digestion of plasmid DNA. Upon receipt at the Special Microbiology Laboratory, each isolate
12, 15, 16); however, Staphylococcus aureus is also important, and the overall incidence of S. aureus peritonitis is approximately 20% in this patient population (3, 9, 13). Regardless of the etiologic agent, the route of infection is assumed to be the intraperitoneal introduction of the infecting organism either directly through the lumen or down the external surface of the intraperitoneal catheter. The source of the infecting organism in patients on CAPD is a matter of debate (3, 13). Sewell et al. (14), Davies et al. (3), and Zimmerman et al. (S. W. Zimmerman, E. Ahrens, C. A. Johnson, W. Craig, J. Leggett, M. O'Brien, L. Oxton, E. Roecker, and S. Engeseth, Abstr. Kidney Int. 37:335,
1990) found that patients on CAPD who were carriers of S. at higher risk of subsequent infection than noncarriers. However, they did not subtype the organisms and, hence, could not prove that the colonizing strains were identical to those isolated from peritoneal fluid. More recently, Luzar et al. (9) reported that in patients who were beginning CAPD, the nasal carriage of S. aureus was associated with an increased risk of catheter-exit-site infection and peritonitis. Furthermore, in 85% of the patients with clinical S. aureus infections, the strain from the nares and the strain causing the infection were similar in their phage types and antibiotic profiles. The present study was designed to define further the relationship between colonizing and infecting strains of S. aureus. We obtained surveillance and peritonitis-associated strains of S. aureus, which we subsequently typed by restriction endonuclease digestion of plasmid DNA. aureus were
*
Corresponding author. 1898
S. AUREUS COLONIZATION IN CAPD
VOL. 28, 1990
was passaged twice on blood agar and stored frozen at -20°C in skim milk. Restriction endonuclease analysis of plasmid DNA. Stored isolates of S. aureus were plated onto blood agar and incubated at 35°C for 48 h. Multiple colonies were removed from the plate with a sterile swab, inoculated into 10 ml of tryptic soy broth, and incubated overnight at 35°C. Organisms were lysed and DNA was extracted by the method of Nahaie et al. (10). Restriction endonuclease digestion of plasmid DNA was performed with EcoRI (New England BioLabs, Inc., Boston, Mass.) according to the instructions of the manufacturer. DNA was digested at 37°C for 2 h, and the restriction fragments were separated by electrophoresis on 0.7% agarose gels containing ethidium bromide. Electrophoresis was performed with Tris borate-EDTA buffer at 100 V for 4 h. Molecular weight markers were included in each gel. Gels were photographed by using UV light, and the restriction digest patterns of the various isolates were compared. DNA from isolates with similar, but not identical, EcoRI patterns were run on a new gel following digestion with either HindIII or EcoRI. Strains were considered to be the same if identical patterns were observed after digestion with either enzyme.
RESULTS Of the 43 patients studied, two or more surveillance or diagnostic (peritoneal fluid) cultures of samples from 39 of them yielded S. aureus. A total of 50 episodes of peritonitis in 35 patients were diagnosed during the study period, including 16 episodes (15 patients) caused by S. aureus. A total of 117 isolates of S. aureus from 30 patients were available for typing, including 51 isolates (22 patients) from the nares, 58 isolates (27 patients) from pericatheter skin, and 8 isolates (six patients) from peritoneal fluid, from patients with peritonitis (Table 1). We observed 28 different restriction endonuclease subtypes among the 117 isolates (Table 1). Figures 1 to 3 show examples of nine distinct subtypes obtained after digestion with EcoRI (Fig. 1 and 2) or HindIII (Fig. 3). Of the 28 restriction endonuclease subtypes, 20 (67%) were found in isolates from single patients. Only subtypes X (four patients; Figure 1, lane 2) and BB (nine patients; Fig. 1, lanes 3 and 7 to 9) were observed in more than two patients. To determine whether patients were persistently colonized with the same strain (subtype) of S. aureus, we compared the restriction endonuclease digest patterns of isolates in samples from the nares (15 patients), pericatheter skin (18 patients), or paired nares and pericatheter skin (20 patients) that, on culturing, were positive at least twice. Although 80% of the patients with nasal colonization and 67% of the patients with pericatheter skin colonization were colonized by more than one distinct subtype of S. aureus, in 60 and 72%, respectively, S. aureus isolates with identical restriction endonuclease digestion patterns were recovered on more than one occasion. Likewise, in 95% of the patients in whom S. aureus was isolated from both the nares and pericatheter skin, the organisms from both sites had the same restriction endonuclease subtype. Examples of repeated isolation of identical subtypes from nasal and/or pericatheter skin surveillance samples are provided in Fig. 1 to 3. Although a total of 16 episodes of S. aureus peritonitis were observed during the study period, colonizing and infecting isolates were available from only six patients (7 episodes). The remaining nine episodes of S. aureus perito-
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TABLE 1. Restriction endonuclease subtypes
of S. aureus isolates Restriction endonuclease subtype
Total no. of isolates (no. of patients)
No plasmid A B C D E F G H
17 (10) 1(1) 2 (1) 1 (1) 4 (2) 1(1) 7 (1) 2 (2) 3 (1) 3 (1) 4(1) 1(1) 2 (1) 2 (1) 7 (2) 6(1) 1 (1) 1 (1) 4 (2) 4 (1) 1(1) 1 (1) 4 (2)
K N
K L M N S
P
Q R S T U V W X Y z AA BB
No. of isolates (no. of patients) from each site Nares
Pericatheter skin
5 (5) 1(1) 1 (1) 1 (1) 1(1)
12 (8)
3 (1) 1(1) 1(1) 1(1) 2(1) 1(1) 1(1) 1(1) 5 (2) 2(1) 1 (1) 1 (1) 1(1) 2 (1)
1 (1)
3 (2) 1(1) 3 (1) 1(1) 2 (1) 2 (1) 2 (1)
1 (1)
1(1) 1(1) 2 (1) 3 (1)
1 (1)
3 (2) 2 (1)
1(1) 1()
2 (2)
1 (1)
7 (4) 2 (1) 1 (1) 9 (2) 18 (9)
Peritoneal fluid
3 1 1 5 8
(2) (1) (1)
4 (4) 1 (1)
(2) (7)
4 (2) 7 (6)
2 (2) 1 (1)
3 (2)
nitis all occurred in patients with nasal or skin colonization with S. aureus; however, these isolates were unavailable for typing. To determine whether the strains of S. aureus that caused peritonitis were identical to those that colonized patients on CAPD, we compared the restriction endonuclease digest patterns of 8 peritoneal fluid isolates, 13 nasal isolates, and 13 pericatheter skin isolates from six patients with peritonitis (Table 2). These isolates constituted all of the S. aureus isolates obtained from the six infected patients. In each case, the same restriction endonuclease subtype was isolated from both the peritoneal fluid and at least one surveillance site. The same restriction endonuclease subtype was isolated from all cultures and on multiple occasions in three patients (patients 2, 7, and 22). An example of isolation of subtype BB from peritoneal fluid, nares, and pericatheter sites is provided in Fig. 1 (lanes 7 to 9). One patient (patient 27) had two distinct episodes of peritonitis, each caused by a different subtype, only one of which (subtype V) was isolated from a surveillance site. Two patients (patients 28 and 29) were colonized with other subtypes, in addition to the subtype isolated from peritoneal fluid. DISCUSSION The relationship between nasal and skin colonization with S. aureus and subsequent infection has been well established by Yu et al. (17) for patients on hemodialysis. Those investigators found that patients on hemodialysis were colonized persistently by the same phage type of S. aureus and that the colonizing strain was also implicated in the infection. Until recently, such data were lacking for patients on
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PIGNATARI ET AL. 1
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FIG. 1. Examples of similarities and differences among strains from four patients following restriction endonuclease digestion of plasmid DNA with EcoRI. Lanes 1 and 2, Pericatheter (subtype S) and nasal (subtype X) isolates, respectively, from patient 20; lanes 3 and 4, pericatheter (subtype BB) and nasal (subtype K) isolates, respectively, from patient 9; lanes 5 and 6, pericatheter and nasal isolates (both subtype t), respectively, from patient 1; lanes 7, 8, and 9, peritoneal fluid, pericatheter, and nasal isolates (all subtype BB), respectively, from patient 7; lane 10, molecular weight standard of lambda phage DNA digested with HindIII.
9 10
FIG. 2. Agarose gel electrophoresis of EcoRI-digested plasmid DNA from three patients. Lanes 1 to 3, Peritoneal fluid (subtype V), pericatheter (subtype AA), and nasal (subtype AA) isolates, respectively, from patient 29; lane 4, molecular weight standard of lambda phage DNA digested with HindIII; lanes 5 to 7, three pericatheter isolates (all subtype O) from patient 28; lanes 8 to 10, three nasal isolates (all subtype F) from patient 22. In each case the EcoRI patterns of the isolates from the same patient were similar, but not identical, and required further analysis with HindIII in order to clarify whether the isolates were of the same or different subtypes
(see Fig. 3).
CAPD. Although Sewell et al. (14), Davies et al. (3), and Zimmerman et al. (Zimmerman et al., Abstr. Kidney Int. 37:335, 1990) suggested that nasal and skin colonization with S. aureus was a risk factor for subsequent peritonitis in patients on CAPD, they did not identify specific strains or subspecies. A recent study by Luzar et al. (9) confirmed these findings and also used phage typing to document the fact that the strain of S. aureus that colonized the nares was frequently the same strain that caused infection in patients on CAPD. Eisenberg et al. (4) used plasmid pattern analysis to study strain variation among colonizing and infecting isolates of Staphylococcus epidermidis obtained from patients on CAPD. They found that serial surveillance isolates usually were different strains and that peritonitis-associated isolates were frequently different from surveillance isolates. Results of these studies emphasize the need to identify specific strains within species of staphylococci in studies that attempt to examine the persistence of colonization and the relationship between colonization and infection. In the present study, 28 of 43 (65%) patients on CAPD in Brazil were persistently colonized (two or more positive surveillance cultures) at one or more sites with S. aureus. This high colonization rate is similar to that reported by Yu et al. (17) for patients on hemodialysis and is considerably higher than that observed by Sewell et al. (14) for patients on CAPD (33%). Our data demonstrate considerable strain variation among serial isolates of S. aureus obtained from nasal and pericatheter skin surveillance sites. A total of 80% of the patients with nasal colonization and 67% of the patients with pericatheter skin colonization were colonized at different times with isolates of S. aureus that had at least two distinct restriction endonuclease subtypes. In general, patients did not share S. aureus isolates with the same restriction endonuclease subtypes; however, isolates with two distinct subtypes, X and BB, were shared by four and
nine patients, respectively. Although these results raise the possibility of limited patient-to-patient transmission of these subtypes, horizontal transmission of S. aureus was not the focus of this study and was not investigated further. Despite the variations in restriction endonuclease subtypes, our results are similar to those of Yu et al. (17), in that 1
2
3
4
5
6
7
8
9
10
FIG. 3. Agarose gel electrophoresis of HindIII-digested plasmid DNA from patients 22, 28, and 29 (see Fig. 2). Lanes 1 to 3, Nasal (subtype AA), pericatheter (subtype AA), and peritoneal fluid (subtype V) isolates, respectively, from patient 29; lane 4, molecular weight standards (HindIII-digested lambda phage DNA); lanes 5 to 7, three pericatheter isolates (all subtype O) from patient 28; lanes 8 to 10, three nasal isolates (all subtype F) from patient 22. Digestion with HindIII confirmed the similarities and differences among the isolates from all three patients.
S. AUREUS COLONIZATION IN CAPD
VOL. 28, 1990
TABLE 2. Relationship among isolates of S. aureus from peritoneal fluid, nares, and pericatheter skin Patient no.
Restriction endonuclease subtype (no. of times isolated) Peritoneal fluid
2 7 22 27 28 29
BB BB (2) F V, W 0 V
Nares
BB (2) F (3)
0 (2), Z AA (4), U
Pericatheter skin
BB BB F (3) V 0 (3) AA (3), V
in 60% of the patients with nasal colonization and 72% of the patients with pericatheter skin colonization, isolates with a given subtype were recovered on more than one occasion. Furthermore, the majority of patients (95%) with both nasal and pericatheter skin colonization were colonized with isolates with the same subtypes at both sites. Although the number of infected patients was small, 100% of those with peritonitis were infected with a subtype that was isolated from the nasal and/or pericatheter skin surveillance samples. A potential limitation of this study is that the increased prevalence of subtype BB (18 of 117 isolates) may skew the results in favor of isolating organisms with the same subtype from multiple strains. This would be more of a problem if we were concerned with horizontal transmission. The large number of subtypes that we identified and the fact that four of the six patients with peritonitis were colonized and infected with organisms containing subtypes other than BB suggests that this is not a major concern. Thus, our results with patients on CAPD support the conclusions of Yu et al. (17) and Luzar et al. (9) that patients on dialysis who develop infections with S. aureus become infected with endogenous (colonizing) strains. In the present study, we obtained excellent strain discrimination by using restriction endonuclease digestion of plasmid DNA. Although bacteriophage typing has been the most common used epidemiologic typing system for S. aureus, several studies have show that plasmid pattern analysis (1, 2) and restriction endonuclease analysis of plasmid DNA (7, 11) are superior in terms of typability, reproducibility, strain discrimination, and ease of performance. On the other hand, Gaston et al. (5) reported that phage typing, whole-cell protein profiles, and immunoblotting profiles all provide better strain discrimination than does plasmid pattern analysis of methicillin-resistant S. aureus. However, they did not perform restriction endonuclease digestion of the plasmid DNA, which may be essential to the analysis of plasmid profiles of methicillin-resistant and methicillin-susceptible S. aureus (7, 18). Given these findings and the fact that S. aureus is the most frequent cause of peritonitis in patients on CAPD in Brazil (13), prophylactic measures designed to eradicate the carrier state appear to be warranted. The efficacy of this approach was documented by Yu et al. (17) in patients on hemodialysis who demonstrated that oral rifampin prophylaxis eradicates the carrier state and significantly decreases the incidence of S. aureus infection. Furthermore, a new topical agent, mupirocin (Beecham Laboratories, Bristol, Tenn.), which eradicates both nasal and hand carriage of S. aureus (8), has been reported to decrease S. aureus infections in patients on hemodialysis (J. R. Boelaert, Y. DeBaere, C. Godard, H. W. VanLanduyt, and A. Z. St. Jan Brugge, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no.
1901
1262, 1989). We feel that mupirocin should be investigated further in clinical trials with patients on CAPD. In summary, we used restriction endonuclease analysis of plasmid DNA to document the diversity of strains of S. aureus that colonize and infect patients on CAPD in Brazil. In the majority of patients, nasal and/or pericatheter skin isolates with identical restriction endonuclease digest patterns were recovered on more than one occasion, and 95% of patients with both nasal and pericatheter skin colonization were colonized with organisms with the same subtypes at both sites. Furthermore, 100% of the patients with peritonitis were infected with an S. aureus subtype which colonized the nares, pericatheter skin, or both. Thus, our data illustrate that restriction endonuclease analysis of plasmid DNA is a useful method for subtyping S. aureus. Our results also demonstrate the importance of an endogenous source in the development of CAPD-associated peritonitis. Studies designed to evaluate whether chemophorphylaxis eliminates colonization and prevents infection in patients on CAPD are warranted and should include the newer topical antistaphylococcal agents. ACKNOWLEDGMENTS We acknowledge the excellent secretarial skills of Ruth Kjaer. This work was supported in part by the Department of Veterans Affairs. A.P. is a Visiting Research Scientist in the Department of Pathology, University of Iowa College of Medicine, and is supported by grant 204480/88-4/CL from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo, Brazil. LITERATURE CITED 1. Archer, G. L., and C. G. Mayhall. 1983. Comparison of epidemiological markers used in the investigation of an outbreak of methicillin-resistant Staphylococcus aureus infections. J. Clin. Microbiol. 18:395-399. 2. Collins, J. K., J. S. Smith, and M. T. Kelly. 1984. Comparison of phage typing, plasmid mapping, and antibiotic resistance patterns as epidemiologic markers in a nosocomial outbreak of
methicillin-resistant Staphylococcus aureus infections. Diagn. Microbiol. Infect. Dis. 2:233-245. 3. Davies, S. J., C. S. Ogg, J. S. Cameron, S. Poston, and W. C. Noble. 1989. Staphylococcus aureus nasal carriage, exit-site infection and catheter loss in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Peritoneal Dialysis Int. 9:61-64. 4. Eisenberg, E. S., M. Ambalu, G. Sylagi, V. Aning, and R. Soeiro. 1987. Colonization of skin and development of peritonitis due to coagulase-negative staphylococci in patients undergoing peritoneal dialysis. J. Infect. Dis. 156:478-482. 5. Gaston, M. S., P. S. Duif, J. Naidoo, K. Ellis, J. I. S. Roberts, J. F. Richardson, R. R. Marples, and E. M. Cooke. 1988. Evaluation of electrophoretic methods for typing methicillinresistant Staphylococcus aureus. J. Med. Microbiol. 26:189197. 6. Gruer, L. D., R. Bartlett, and G. A. J. Ayliffe. 1984. Species identification and antibiotic sensitivity of coagulase-negative staphylococci from CAPD peritonitis. J. Antimicrob. Chemother. 13:577-583. 7. Hartstein, A. I., V. H. Morthland, S. Eng, G. L. Archer, F. D. Schoenknecht, and A. L. Rashad. 1989. Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates. J. Clin. Microbiol. 27:1874-1879. 8. Hill, R. L. R., G. J. Duckworth, and M. W. Casewell. 1988. Elimination of nasal carriage of methicillin-resistant Staphylococcus aureus with mupirocin during a hospital outbreak. J. Antimicrob. Chemother. 22:377-384. 9. Luzar, M. A., G. A. Coles, B. Faller, A. Slingeneyer, G. DahDah, C. Briat, C. Wone, Y. Knefati, M. Kessler, and F. Peluso. 1990. Staphylococcus aureus nasal carriage and infection in patients
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12.
13.
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on continuous ambulatory peritoneal dialysis. N. Engl. J. Med. 322:505-509. Nahaie, M. R., M. Goodfellow, and C. R. Harwood. 1984. A rapid screening procedure for staphylococcal plasmids. J. Microbiol. Methods 2:73-81. Rhinehart, E., D. M. Shlaes, T. F. Keys, J. Serkey, IP. Kirkley, C. Kim, C. A. Currie-McCumber, and G. Hall. 1987. Nosocomial clonal dissemination of methicillin-resistant Staphylococcus aureus. Arch. Intern. Med. 147:521-524. Rubin, J., W. A. Rogers, H. M. Taylor, E. D. Everett, B. F. Prowant, L. V. Fruto, and K. D. Nolph. 1980. Peritonitis during continuous ambulatory peritoneal dialysis. Ann. Intern. Med. 92:7-13. Sesso, R., S. Draibe, A. Castelo, I. Sato, I. Leme, D. Barbosa, and O. Ramos. 1989. Staphylococcus aureus skin carriage and development of peritonitis in patients on continuous ambulatory peritoneal dialysis. Clin. Nephrol. 31:264-268.
J. CLIN. MICROBIOL. 14. Sewell, C. M., J. Clarridge, C. Lacke, E. J. Weinman, and E. J. Young. 1982. Staphylococcal nasal carriage and subsequent infection in peritoneal dialysis patients. J. Am. Med. Assoc. 248:1493-1495. 15. Vas, S. I. 1983. Microbiologic aspects of chronic ambulatory peritoneal dialysis. Kidney Int. 23:83-92. 16. West, T. E., J. J. Walsh, C. P. Krol, and D. Amsterdam. 1986. Staphylococcal peritonitis in patients on peritoneal dialysis. J. Clin. Microbiol. 23:809-812. 17. Yu, V. L., A. Goetz, M. Wagener, P. B. Smith, J. D. Rihs, J. Hanchett, and J. J. Zuravleff. 1986. Staphylococcus aureus nasal carriage and infection in patients on hemodialysis. N. Engl. J. Med. 315:91-96. 18. Zuccarelli, A. J., I. Roy, G. P. Harding, and J. J. Couperus. 1990. Diversity and stability of restriction enzyme profiles of plasmid DNA from methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 28:97-102.
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1990, p. 1898-1902
0095-1137/90/091898-05$02.00/0 Copyright © 1990, American Society for Microbiology
Staphylococcus aureus Colonization and Infection in Patients Continuous Ambulatory Peritoneal Dialysis
on
A. PIGNATARI,"12 M. PFALLER,l13* R. HOLLIS,' R. SESSO,4 I. LEME,2 AND L. HERWALDT5 Departments of Pathology' and Internal Medicine,5 University of lowa College of Medicine, Iowa City, Iowa 52242; Department of Veterans Affairs Medical Center, Iowa City, Iowa 522403; and Disciplina de Doencas Infecciosas e Parasitarias2 and Disciplina de Nefrologia,4 Escola Paulista de Medicina, Sao Paulo, Brazil Received 22 February 1990/Accepted 11 June 1990
Staphylococcus aureus is the most common cause of peritonitis in patients undergoing peritoneal dialysis in Brazil. Using restriction endonuclease analysis of plasmid DNA, we investigated the importance of chronic carriage of S. aureus in the development of peritonitis in patients on continuous ambulatory peritoneal dialysis at the Division of Nephrology, Escola Paulista de Medicina, Sao Paulo, Brazil. A total of 117 isolates (30 patients) of S. aureus were available for typing, including 51 isolates (22 patients) from the nares, 58 isolates (27 patients) from pericatheter skin, and 8 isolates (6 patients) from peritoneal fluid, from patients with peritonitis. Restriction endonuclease subtyping showed that although most patients harbored more than one subtype of S. aureus, in the majority of patients nasal and/or pericatheter skin isolates with identical restriction endonuclease digest patterns were recovered on more than one occasion. Furthermore, 95% of patients with both nasal and pericatheter colonization were colonized with the same subtypes at both sites. All of the patients with peritonitis were infected with a subtype which colonized the nares, pericatheter skin, or both. These results demonstrate the importance of an endogenous source of S. aureus in the development of continuous ambulatory peritoneal dialysis-associated peritonitis. MATERIALS AND METHODS
Peritonitis is a major complication in patients on continuous ambulatory peritoneal dialysis (CAPD) (3, 4, 9, 12-14). In many countries, the most common cause of peritonitis in patients on CAPD is coagulase-negative staphylococci (4, 6,
Patients. All 43 patients on CAPD at the Division of Nephrology, Escola Paulista de Medicina (Sao Paulo, Brazil), from April 1987 through June 1988 were included in the surveillance study. The clinical characteristics of these patients have been described previously (13). Surveillance samples from the nares and pericatheter skin were obtained every 2 months (sampling period). Samples of peritoneal dialysis fiuid were also cultured when it was clinically indicated for the diagnosis of peritonitis. Only patients with at least two samples that were positive for S. aureus were included in the present study. Definitions. Peritonitis was defined as cloudy dialysis effluent containing more than 100 leukocytes per mm3, with more than 50% of these cells being polymorphonuclear leukocytes. The presence of an organism on Gram staining or a positive culture was confirmatory but not necessary for the diagnosis of peritonitis. Microbiology. Surveillance samples of the nares and pericatheter skin were obtained by using sterile, premoistened calcium alginate swabs (Cefar-Farmaco Diagnostic Ltda., Sao Paulo, Brazil) and were transported to the microbiology laboratory, where they were immediately streaked onto plates containing tryptic soy agar with 5% sheep blood, MacConkey agar, and mannitol-salt agar (Difco Laboratories, Detroit, Mich.). If patients developed peritonitis, 5 ml of peritoneal fluid was inoculated into each of two bottles (Hemo-cult 1; Laborclin Ltda., Parana, Brazil) containing 50 ml of tryptic soy broth (Difco). All cultures were incubated at 36°C for 48 h and examined daily for evidence of growth. Gram-positive cocci which produced catalase and coagulase were identified as S. aureus. Isolates of S. aureus were saved on nutrient agar slants and sent to the Special Microbiology Laboratory at the University of Iowa for typing by restriction endonuclease digestion of plasmid DNA. Upon receipt at the Special Microbiology Laboratory, each isolate
12, 15, 16); however, Staphylococcus aureus is also important, and the overall incidence of S. aureus peritonitis is approximately 20% in this patient population (3, 9, 13). Regardless of the etiologic agent, the route of infection is assumed to be the intraperitoneal introduction of the infecting organism either directly through the lumen or down the external surface of the intraperitoneal catheter. The source of the infecting organism in patients on CAPD is a matter of debate (3, 13). Sewell et al. (14), Davies et al. (3), and Zimmerman et al. (S. W. Zimmerman, E. Ahrens, C. A. Johnson, W. Craig, J. Leggett, M. O'Brien, L. Oxton, E. Roecker, and S. Engeseth, Abstr. Kidney Int. 37:335,
1990) found that patients on CAPD who were carriers of S. at higher risk of subsequent infection than noncarriers. However, they did not subtype the organisms and, hence, could not prove that the colonizing strains were identical to those isolated from peritoneal fluid. More recently, Luzar et al. (9) reported that in patients who were beginning CAPD, the nasal carriage of S. aureus was associated with an increased risk of catheter-exit-site infection and peritonitis. Furthermore, in 85% of the patients with clinical S. aureus infections, the strain from the nares and the strain causing the infection were similar in their phage types and antibiotic profiles. The present study was designed to define further the relationship between colonizing and infecting strains of S. aureus. We obtained surveillance and peritonitis-associated strains of S. aureus, which we subsequently typed by restriction endonuclease digestion of plasmid DNA. aureus were
*
Corresponding author. 1898
S. AUREUS COLONIZATION IN CAPD
VOL. 28, 1990
was passaged twice on blood agar and stored frozen at -20°C in skim milk. Restriction endonuclease analysis of plasmid DNA. Stored isolates of S. aureus were plated onto blood agar and incubated at 35°C for 48 h. Multiple colonies were removed from the plate with a sterile swab, inoculated into 10 ml of tryptic soy broth, and incubated overnight at 35°C. Organisms were lysed and DNA was extracted by the method of Nahaie et al. (10). Restriction endonuclease digestion of plasmid DNA was performed with EcoRI (New England BioLabs, Inc., Boston, Mass.) according to the instructions of the manufacturer. DNA was digested at 37°C for 2 h, and the restriction fragments were separated by electrophoresis on 0.7% agarose gels containing ethidium bromide. Electrophoresis was performed with Tris borate-EDTA buffer at 100 V for 4 h. Molecular weight markers were included in each gel. Gels were photographed by using UV light, and the restriction digest patterns of the various isolates were compared. DNA from isolates with similar, but not identical, EcoRI patterns were run on a new gel following digestion with either HindIII or EcoRI. Strains were considered to be the same if identical patterns were observed after digestion with either enzyme.
RESULTS Of the 43 patients studied, two or more surveillance or diagnostic (peritoneal fluid) cultures of samples from 39 of them yielded S. aureus. A total of 50 episodes of peritonitis in 35 patients were diagnosed during the study period, including 16 episodes (15 patients) caused by S. aureus. A total of 117 isolates of S. aureus from 30 patients were available for typing, including 51 isolates (22 patients) from the nares, 58 isolates (27 patients) from pericatheter skin, and 8 isolates (six patients) from peritoneal fluid, from patients with peritonitis (Table 1). We observed 28 different restriction endonuclease subtypes among the 117 isolates (Table 1). Figures 1 to 3 show examples of nine distinct subtypes obtained after digestion with EcoRI (Fig. 1 and 2) or HindIII (Fig. 3). Of the 28 restriction endonuclease subtypes, 20 (67%) were found in isolates from single patients. Only subtypes X (four patients; Figure 1, lane 2) and BB (nine patients; Fig. 1, lanes 3 and 7 to 9) were observed in more than two patients. To determine whether patients were persistently colonized with the same strain (subtype) of S. aureus, we compared the restriction endonuclease digest patterns of isolates in samples from the nares (15 patients), pericatheter skin (18 patients), or paired nares and pericatheter skin (20 patients) that, on culturing, were positive at least twice. Although 80% of the patients with nasal colonization and 67% of the patients with pericatheter skin colonization were colonized by more than one distinct subtype of S. aureus, in 60 and 72%, respectively, S. aureus isolates with identical restriction endonuclease digestion patterns were recovered on more than one occasion. Likewise, in 95% of the patients in whom S. aureus was isolated from both the nares and pericatheter skin, the organisms from both sites had the same restriction endonuclease subtype. Examples of repeated isolation of identical subtypes from nasal and/or pericatheter skin surveillance samples are provided in Fig. 1 to 3. Although a total of 16 episodes of S. aureus peritonitis were observed during the study period, colonizing and infecting isolates were available from only six patients (7 episodes). The remaining nine episodes of S. aureus perito-
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TABLE 1. Restriction endonuclease subtypes
of S. aureus isolates Restriction endonuclease subtype
Total no. of isolates (no. of patients)
No plasmid A B C D E F G H
17 (10) 1(1) 2 (1) 1 (1) 4 (2) 1(1) 7 (1) 2 (2) 3 (1) 3 (1) 4(1) 1(1) 2 (1) 2 (1) 7 (2) 6(1) 1 (1) 1 (1) 4 (2) 4 (1) 1(1) 1 (1) 4 (2)
K N
K L M N S
P
Q R S T U V W X Y z AA BB
No. of isolates (no. of patients) from each site Nares
Pericatheter skin
5 (5) 1(1) 1 (1) 1 (1) 1(1)
12 (8)
3 (1) 1(1) 1(1) 1(1) 2(1) 1(1) 1(1) 1(1) 5 (2) 2(1) 1 (1) 1 (1) 1(1) 2 (1)
1 (1)
3 (2) 1(1) 3 (1) 1(1) 2 (1) 2 (1) 2 (1)
1 (1)
1(1) 1(1) 2 (1) 3 (1)
1 (1)
3 (2) 2 (1)
1(1) 1()
2 (2)
1 (1)
7 (4) 2 (1) 1 (1) 9 (2) 18 (9)
Peritoneal fluid
3 1 1 5 8
(2) (1) (1)
4 (4) 1 (1)
(2) (7)
4 (2) 7 (6)
2 (2) 1 (1)
3 (2)
nitis all occurred in patients with nasal or skin colonization with S. aureus; however, these isolates were unavailable for typing. To determine whether the strains of S. aureus that caused peritonitis were identical to those that colonized patients on CAPD, we compared the restriction endonuclease digest patterns of 8 peritoneal fluid isolates, 13 nasal isolates, and 13 pericatheter skin isolates from six patients with peritonitis (Table 2). These isolates constituted all of the S. aureus isolates obtained from the six infected patients. In each case, the same restriction endonuclease subtype was isolated from both the peritoneal fluid and at least one surveillance site. The same restriction endonuclease subtype was isolated from all cultures and on multiple occasions in three patients (patients 2, 7, and 22). An example of isolation of subtype BB from peritoneal fluid, nares, and pericatheter sites is provided in Fig. 1 (lanes 7 to 9). One patient (patient 27) had two distinct episodes of peritonitis, each caused by a different subtype, only one of which (subtype V) was isolated from a surveillance site. Two patients (patients 28 and 29) were colonized with other subtypes, in addition to the subtype isolated from peritoneal fluid. DISCUSSION The relationship between nasal and skin colonization with S. aureus and subsequent infection has been well established by Yu et al. (17) for patients on hemodialysis. Those investigators found that patients on hemodialysis were colonized persistently by the same phage type of S. aureus and that the colonizing strain was also implicated in the infection. Until recently, such data were lacking for patients on
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FIG. 1. Examples of similarities and differences among strains from four patients following restriction endonuclease digestion of plasmid DNA with EcoRI. Lanes 1 and 2, Pericatheter (subtype S) and nasal (subtype X) isolates, respectively, from patient 20; lanes 3 and 4, pericatheter (subtype BB) and nasal (subtype K) isolates, respectively, from patient 9; lanes 5 and 6, pericatheter and nasal isolates (both subtype t), respectively, from patient 1; lanes 7, 8, and 9, peritoneal fluid, pericatheter, and nasal isolates (all subtype BB), respectively, from patient 7; lane 10, molecular weight standard of lambda phage DNA digested with HindIII.
9 10
FIG. 2. Agarose gel electrophoresis of EcoRI-digested plasmid DNA from three patients. Lanes 1 to 3, Peritoneal fluid (subtype V), pericatheter (subtype AA), and nasal (subtype AA) isolates, respectively, from patient 29; lane 4, molecular weight standard of lambda phage DNA digested with HindIII; lanes 5 to 7, three pericatheter isolates (all subtype O) from patient 28; lanes 8 to 10, three nasal isolates (all subtype F) from patient 22. In each case the EcoRI patterns of the isolates from the same patient were similar, but not identical, and required further analysis with HindIII in order to clarify whether the isolates were of the same or different subtypes
(see Fig. 3).
CAPD. Although Sewell et al. (14), Davies et al. (3), and Zimmerman et al. (Zimmerman et al., Abstr. Kidney Int. 37:335, 1990) suggested that nasal and skin colonization with S. aureus was a risk factor for subsequent peritonitis in patients on CAPD, they did not identify specific strains or subspecies. A recent study by Luzar et al. (9) confirmed these findings and also used phage typing to document the fact that the strain of S. aureus that colonized the nares was frequently the same strain that caused infection in patients on CAPD. Eisenberg et al. (4) used plasmid pattern analysis to study strain variation among colonizing and infecting isolates of Staphylococcus epidermidis obtained from patients on CAPD. They found that serial surveillance isolates usually were different strains and that peritonitis-associated isolates were frequently different from surveillance isolates. Results of these studies emphasize the need to identify specific strains within species of staphylococci in studies that attempt to examine the persistence of colonization and the relationship between colonization and infection. In the present study, 28 of 43 (65%) patients on CAPD in Brazil were persistently colonized (two or more positive surveillance cultures) at one or more sites with S. aureus. This high colonization rate is similar to that reported by Yu et al. (17) for patients on hemodialysis and is considerably higher than that observed by Sewell et al. (14) for patients on CAPD (33%). Our data demonstrate considerable strain variation among serial isolates of S. aureus obtained from nasal and pericatheter skin surveillance sites. A total of 80% of the patients with nasal colonization and 67% of the patients with pericatheter skin colonization were colonized at different times with isolates of S. aureus that had at least two distinct restriction endonuclease subtypes. In general, patients did not share S. aureus isolates with the same restriction endonuclease subtypes; however, isolates with two distinct subtypes, X and BB, were shared by four and
nine patients, respectively. Although these results raise the possibility of limited patient-to-patient transmission of these subtypes, horizontal transmission of S. aureus was not the focus of this study and was not investigated further. Despite the variations in restriction endonuclease subtypes, our results are similar to those of Yu et al. (17), in that 1
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FIG. 3. Agarose gel electrophoresis of HindIII-digested plasmid DNA from patients 22, 28, and 29 (see Fig. 2). Lanes 1 to 3, Nasal (subtype AA), pericatheter (subtype AA), and peritoneal fluid (subtype V) isolates, respectively, from patient 29; lane 4, molecular weight standards (HindIII-digested lambda phage DNA); lanes 5 to 7, three pericatheter isolates (all subtype O) from patient 28; lanes 8 to 10, three nasal isolates (all subtype F) from patient 22. Digestion with HindIII confirmed the similarities and differences among the isolates from all three patients.
S. AUREUS COLONIZATION IN CAPD
VOL. 28, 1990
TABLE 2. Relationship among isolates of S. aureus from peritoneal fluid, nares, and pericatheter skin Patient no.
Restriction endonuclease subtype (no. of times isolated) Peritoneal fluid
2 7 22 27 28 29
BB BB (2) F V, W 0 V
Nares
BB (2) F (3)
0 (2), Z AA (4), U
Pericatheter skin
BB BB F (3) V 0 (3) AA (3), V
in 60% of the patients with nasal colonization and 72% of the patients with pericatheter skin colonization, isolates with a given subtype were recovered on more than one occasion. Furthermore, the majority of patients (95%) with both nasal and pericatheter skin colonization were colonized with isolates with the same subtypes at both sites. Although the number of infected patients was small, 100% of those with peritonitis were infected with a subtype that was isolated from the nasal and/or pericatheter skin surveillance samples. A potential limitation of this study is that the increased prevalence of subtype BB (18 of 117 isolates) may skew the results in favor of isolating organisms with the same subtype from multiple strains. This would be more of a problem if we were concerned with horizontal transmission. The large number of subtypes that we identified and the fact that four of the six patients with peritonitis were colonized and infected with organisms containing subtypes other than BB suggests that this is not a major concern. Thus, our results with patients on CAPD support the conclusions of Yu et al. (17) and Luzar et al. (9) that patients on dialysis who develop infections with S. aureus become infected with endogenous (colonizing) strains. In the present study, we obtained excellent strain discrimination by using restriction endonuclease digestion of plasmid DNA. Although bacteriophage typing has been the most common used epidemiologic typing system for S. aureus, several studies have show that plasmid pattern analysis (1, 2) and restriction endonuclease analysis of plasmid DNA (7, 11) are superior in terms of typability, reproducibility, strain discrimination, and ease of performance. On the other hand, Gaston et al. (5) reported that phage typing, whole-cell protein profiles, and immunoblotting profiles all provide better strain discrimination than does plasmid pattern analysis of methicillin-resistant S. aureus. However, they did not perform restriction endonuclease digestion of the plasmid DNA, which may be essential to the analysis of plasmid profiles of methicillin-resistant and methicillin-susceptible S. aureus (7, 18). Given these findings and the fact that S. aureus is the most frequent cause of peritonitis in patients on CAPD in Brazil (13), prophylactic measures designed to eradicate the carrier state appear to be warranted. The efficacy of this approach was documented by Yu et al. (17) in patients on hemodialysis who demonstrated that oral rifampin prophylaxis eradicates the carrier state and significantly decreases the incidence of S. aureus infection. Furthermore, a new topical agent, mupirocin (Beecham Laboratories, Bristol, Tenn.), which eradicates both nasal and hand carriage of S. aureus (8), has been reported to decrease S. aureus infections in patients on hemodialysis (J. R. Boelaert, Y. DeBaere, C. Godard, H. W. VanLanduyt, and A. Z. St. Jan Brugge, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no.
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1262, 1989). We feel that mupirocin should be investigated further in clinical trials with patients on CAPD. In summary, we used restriction endonuclease analysis of plasmid DNA to document the diversity of strains of S. aureus that colonize and infect patients on CAPD in Brazil. In the majority of patients, nasal and/or pericatheter skin isolates with identical restriction endonuclease digest patterns were recovered on more than one occasion, and 95% of patients with both nasal and pericatheter skin colonization were colonized with organisms with the same subtypes at both sites. Furthermore, 100% of the patients with peritonitis were infected with an S. aureus subtype which colonized the nares, pericatheter skin, or both. Thus, our data illustrate that restriction endonuclease analysis of plasmid DNA is a useful method for subtyping S. aureus. Our results also demonstrate the importance of an endogenous source in the development of CAPD-associated peritonitis. Studies designed to evaluate whether chemophorphylaxis eliminates colonization and prevents infection in patients on CAPD are warranted and should include the newer topical antistaphylococcal agents. ACKNOWLEDGMENTS We acknowledge the excellent secretarial skills of Ruth Kjaer. This work was supported in part by the Department of Veterans Affairs. A.P. is a Visiting Research Scientist in the Department of Pathology, University of Iowa College of Medicine, and is supported by grant 204480/88-4/CL from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo, Brazil. LITERATURE CITED 1. Archer, G. L., and C. G. Mayhall. 1983. Comparison of epidemiological markers used in the investigation of an outbreak of methicillin-resistant Staphylococcus aureus infections. J. Clin. Microbiol. 18:395-399. 2. Collins, J. K., J. S. Smith, and M. T. Kelly. 1984. Comparison of phage typing, plasmid mapping, and antibiotic resistance patterns as epidemiologic markers in a nosocomial outbreak of
methicillin-resistant Staphylococcus aureus infections. Diagn. Microbiol. Infect. Dis. 2:233-245. 3. Davies, S. J., C. S. Ogg, J. S. Cameron, S. Poston, and W. C. Noble. 1989. Staphylococcus aureus nasal carriage, exit-site infection and catheter loss in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Peritoneal Dialysis Int. 9:61-64. 4. Eisenberg, E. S., M. Ambalu, G. Sylagi, V. Aning, and R. Soeiro. 1987. Colonization of skin and development of peritonitis due to coagulase-negative staphylococci in patients undergoing peritoneal dialysis. J. Infect. Dis. 156:478-482. 5. Gaston, M. S., P. S. Duif, J. Naidoo, K. Ellis, J. I. S. Roberts, J. F. Richardson, R. R. Marples, and E. M. Cooke. 1988. Evaluation of electrophoretic methods for typing methicillinresistant Staphylococcus aureus. J. Med. Microbiol. 26:189197. 6. Gruer, L. D., R. Bartlett, and G. A. J. Ayliffe. 1984. Species identification and antibiotic sensitivity of coagulase-negative staphylococci from CAPD peritonitis. J. Antimicrob. Chemother. 13:577-583. 7. Hartstein, A. I., V. H. Morthland, S. Eng, G. L. Archer, F. D. Schoenknecht, and A. L. Rashad. 1989. Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates. J. Clin. Microbiol. 27:1874-1879. 8. Hill, R. L. R., G. J. Duckworth, and M. W. Casewell. 1988. Elimination of nasal carriage of methicillin-resistant Staphylococcus aureus with mupirocin during a hospital outbreak. J. Antimicrob. Chemother. 22:377-384. 9. Luzar, M. A., G. A. Coles, B. Faller, A. Slingeneyer, G. DahDah, C. Briat, C. Wone, Y. Knefati, M. Kessler, and F. Peluso. 1990. Staphylococcus aureus nasal carriage and infection in patients
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J. CLIN. MICROBIOL. 14. Sewell, C. M., J. Clarridge, C. Lacke, E. J. Weinman, and E. J. Young. 1982. Staphylococcal nasal carriage and subsequent infection in peritoneal dialysis patients. J. Am. Med. Assoc. 248:1493-1495. 15. Vas, S. I. 1983. Microbiologic aspects of chronic ambulatory peritoneal dialysis. Kidney Int. 23:83-92. 16. West, T. E., J. J. Walsh, C. P. Krol, and D. Amsterdam. 1986. Staphylococcal peritonitis in patients on peritoneal dialysis. J. Clin. Microbiol. 23:809-812. 17. Yu, V. L., A. Goetz, M. Wagener, P. B. Smith, J. D. Rihs, J. Hanchett, and J. J. Zuravleff. 1986. Staphylococcus aureus nasal carriage and infection in patients on hemodialysis. N. Engl. J. Med. 315:91-96. 18. Zuccarelli, A. J., I. Roy, G. P. Harding, and J. J. Couperus. 1990. Diversity and stability of restriction enzyme profiles of plasmid DNA from methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 28:97-102.
