JAC Advance Access originally published online on March 21, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):855-864; doi:10.1093/jac/dkl071
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Commensal isolates of methicillin-resistant Staphylococcus epidermidis are also well equipped to produce biofilm on polystyrene surfaces
1 Faculdade de Farmácia, Universidade Federal Fluminense, Niterói, 24241002 RJ, Brazil; 2 Departamento de Microbiologia Médica, Instituto de Microbiologia Prof Paulo de Góes, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco I, Cidade Universitária, Rio de Janeiro, RJ, 21941590, Brazil; 3 Hospital Samaritano, Rio de Janeiro, 22041010 RJ, Brazil
* Corresponding author. Tel: +55-21-22604193; Fax: +55-21-25608528; E-mail: agnes{at}micro.ufrj.br
Received 20 December 2005; returned 25 January 2006; revised 9 February 2006; accepted 14 February 2006
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Objectives: To study biofilm production and to detect icaAD, atlE and aap genes in 137 isolates of methicillin-resistant Staphylococcus epidermidis (MRSE) obtained from healthy individuals from the community (35 isolates), from hospitalized patients at the Antônio Pedro University Hospital (25 isolates) and from individuals from a home-care system (HCS; 77 isolates).
Methods: Biofilm production was determined in vitro using polystyrene inert surfaces. icaAD, atlE and aap genes were detected using PCR. Hybridization experiments were also carried out to confirm some PCR results. Antimicrobial susceptibility testing was carried out using the NCCLS methods.
Results: Although many of the commensal MRSE isolates produced biofilms, the percentage of biofilm producers was significantly higher (P = 0.0107) among hospital isolates (76%) than among isolates from the community (60%) and from the HCS (57%). An association was observed between multiresistance and biofilm production for isolates obtained from healthy individuals from the community and from household contacts from the HCS (P < 0.0001). The concomitant presence of the ica operon and atlE and aap genes was associated with the strong biofilm-producer phenotype (P < 0.0001).
Conclusion: Because many of the commensal MRSE isolates obtained from nares produced biofilms and carried icaAD, aap and atlE genes, biofilms or such genetic elements should not be used as markers for clinical significance. The biofilm environment seems to increase genetic exchanges and hence may contribute to multiresistance phenotypes.
Keywords: MRSE , biofilm , ica , aap , atlE
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Introduction |
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One of the great challenges of modern medicine is the increasing use of invasive medical procedures such as device implantation. Staphylococcus epidermidis is an important bacterial species of the indigenous flora of the skin, nares and other mucosal surfaces of humans.1–3 Biofilm production by S. epidermidis can occur on almost any kind of catheter and in a variety of other medical devices and implants. Once a biofilm is formed, a chronic infection is generally established and in some cases the patient has to undergo surgical intervention for the removal of the implant. Consequently, the extensive use of such indwelling devices has led to the emergence of new routes of infection associated with biofilm production on the surfaces of such foreign bodies.4–6 Species of Staphylococcus, particularly S. epidermidis and S. aureus, are the bacteria most frequently isolated in this infectious process worldwide. Currently, S. epidermidis is a major agent of catheter-associated bloodstream infection (mainly in children) and urinary tract infection, in addition to infections associated with orthopaedic and cardiac prostheses and also peritoneal catheters.5–10
The process of biofilm production in S. epidermidis has not yet been totally clarified but seems to occur in two important steps: adherence to the inert surface and biofilm accumulation. Many bacterial products are thought to be involved in the initial phase of adherence including the AtlE protein, teichoic acid and also staphylococcal adhesins, proteins which play an important role in plasma-coated biomaterial.11–13 AtlE (encoded by the atlE gene) shows homology with the major autolysin of S. aureus.12,13 It is not fully understood how AtlE is able to connect to inert surfaces. However, studies using an atlE mutant showed a reduced primary adherence to polystyrene, reduced in vitro binding to vitronectin and also reduced surface hydrophobicity. In addition, this mutant was not able to produce biofilm.14
In the second phase, bacteria connected directly or indirectly to the surface of the polymer produce and accumulate an extracellular, amorphous and mucoid polysaccharide material: the biofilm. This is thought to be the main mechanism of bacterial adherence to plastic surfaces and of auto-aggregation.15,16 Studies have indicated that the mature biofilm facilitates colonization and persistence of bacteria in the host.4,15 In S. epidermidis, the ica operon (encoding enzymes for the biosynthesis of the polysaccharide intercellular adhesin; PIA) seems to be essential for the production of biofilm.4,15,16 The ica operon is constituted by the genes icaR (the regulatory gene) and icaADBC (biosynthesis genes).17,18 In addition to PIA, proteins also seem to be involved in the accumulation phase. An example is the associated to accumulation protein (AAP) of 140 kDa. It is proposed that AAP plays a role in anchoring PIA to the S. epidermidis cell surface since a mutant carrying an inactivated aap produced PIA that is loosely attached to the bacterial surface.19
The biofilm and the genes involved in its production (especially ica) have been suggested as markers for clinical significance, based on the findings that S. epidermidis isolates collected from infected patients are more able to produce biofilm than commensal S. epidermidis isolates.20,21 However, contradictions exist since other studies have also reported a higher incidence of biofilm production among commensal S. epidermidis.22 In addition, an association between biofilm production and drug multiresistance has been suggested. It was conjectured that bacterial proximity within the biofilm structure might facilitate the horizontal exchange of genetic information, including antimicrobial resistance genes.23 However, this remains a controversial issue since another study has shown no difference in resistance rates between biofilm-producer and non-producer S. epidermidis isolates from blood.24
To clarify these points, we studied the production of biofilms on polystyrene surfaces in populations of methicillin-resistant S. epidermidis (MRSE) isolates obtained from healthy persons, hospitalized patients and individuals from a home-care system (HCS). In addition, we analysed the relationship between biofilm production and the detection of icaAD, atlE and aap genes and the association between biofilm production and multiresistance.
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Materials and methods |
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Sample collection and bacterial identification
MRSE isolates were obtained from nasal swabs of different populations composed of healthy individuals from the community (35 isolates) and individuals associated with an HCS (77 isolates). These 77 MRSE isolates from the HCS were obtained from patients (10 isolates), household contacts (29 isolates) and healthcare workers (38 isolates). The HCS studied is a private organization, located in Rio de Janeiro city, RJ, which provides patients with assistance in their homes. The majority of the patients enrolled are elderly and have co-morbidities requiring skilled support. Nearly 100 patients are assisted each day and 250 new patients are enrolled in this service on a yearly basis. The household contacts were defined as those individuals who stayed at the patient's house for 
8 h per day and for 4 days per week.
The nasal swabs were immersed in TSB (trypticase soy broth) supplemented with NaCl (7.5%) and methicillin (10 mg/L). The culture was incubated at 37°C for 24–48 h. For isolation and identification of S. epidermidis, the culture was streaked on mannitol salt agar and incubated at 37°C for 24 h. In addition, MRSE isolates were also obtained from infected patients at Antônio Pedro University Hospital (HUAP) located in Niterói city, RJ (25 isolates). The isolates were obtained from single samples from different individuals.
Bacteria were identified using automated methods such as VITEK (bioMérieux Brasil S/A, Rio de Janeiro, RJ, Brazil) and the Micro Scan System (Baxter Diagnostic Inc, West Sacramento, CA, USA). After identification, cultures were stored in 20% glycerol at –70°C.
Approval reference number from ethics committee: CONEP/UFF 27.06.01.
Methicillin resistance
Methicillin-resistant isolates were initially identified by inoculating 100 µL of a turbid culture on TSA containing methicillin 25 mg/L.25 Observation of any growth on the methicillin plate after incubation at 35°C for 24–48 h was considered an indication of methicillin resistance. The detection of the mecA gene was carried out using PCR for the confirmation of methicillin resistance.
Antimicrobial susceptibility test
Disc diffusion testing was carried out as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).26 The following antimicrobial agents (Cecon, São Paulo, Brazil) were tested: penicillin 10 U, oxacillin 5 µg, clindamycin 2 µg, erythromycin 15 µg, tetracycline 30 µg, ciprofloxacin 5 µg, trimethoprim/sulfamethoxazole 1.25/23.75 µg, chloramphenicol 30 µg, rifampicin 5 µg, gentamicin 10 µg, teicoplanin 30 µg and vancomycin 30 µg. Linezolid 5 µg and mupirocin 5 µg discs were purchased from Oxoid Brasil Ltda (São Paulo, SP, Brazil).
Biofilm production
Biofilm assays were performed in TSB supplemented with 1% (w/v) glucose using 96-well polystyrene flat-bottom tissue culture plates (Nunclon; Nalge Nunc International, Rochester, NY, USA). All the procedures were performed as described previously,27 except that the optical densities of the culture (ODg) and of the stained biofilm (ODb) were measured at 570 nm.28 Strains of S. epidermidis 70D (a strong biofilm producer) and Staphylococcus pyogenes 75194 (a biofilm non-producer) were used as positive and negative controls, respectively.29 The biofilm unit (BU) was calculated as defined by Amaral et al.29 using the following formula: ODb/ODg. The criterion used for classifying biofilm production was based on the results obtained for the negative control. The calculated BU for S. pyogenes 75194 was 0.091. Thus, the BU value of an isolate should be more than two times the value obtained for the negative control (strain 75194) for it to be considered a biofilm producer. The isolates were classified according to Amaral et al.29 as follows: biofilm non-producers (BU 
0.182), weak biofilm producers (0.182 < BU 0.364), moderate biofilm producers (0.364 < BU 0.728) and strong biofilm producers (BU > 0.728).
PCR-based detection of the mecA, icaAD, atlE and aap genes
The mecA, icaAD, atlE and aap genes were detected using PCR with specific primers (Table 1). The program used for standard PCRs was the same for all sets of primers: initial denaturation at 94°C for 4 min, 25 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, and a final extension at 72°C for 5 min. The amplified DNA was detected using conventional electrophoresis in 1.5% agarose gels.30
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Hybridization experiments
Total genomic DNA was obtained using the phenol–chloroform technique as described previously,30 except that mutanolysin was used to digest the bacterial cell wall. The DNA preparation was denatured by boiling for 5 min. An aliquot of 20 µL (10 µg) of the total DNA was dropped onto a nylon membrane. The membrane was baked at 80°C for 2 h. The icaAD probe was obtained using PCR as described above. Finally, hybridization was carried out using the Gene Imager AlkPhos Direct Labelling and Detection System kit (Amersham Bioscience do Brasil Ltda, São Paulo, SP, Brazil), following the manufacturer's recommendations.
Statistical analysis
The chi-squared test was used to assess the statistical significance for a confidence level of 95% (
= 0.05).
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Results |
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Biofilm production
The majority of the 25 MRSE isolates obtained from HUAP were collected from infected patients (17 isolates) and the remainder from colonization cases (8 isolates). Of the 17 isolates from infected patients, 13 were obtained from catheter-associated bloodstream infections. Of the 25 isolates, 19 (76%) were able to produce biofilm and 9 of these were obtained from catheter-associated bloodstream infections. The MRSE isolates were classified as weak (9 out of 25; 36%), moderate (5 out of 25; 26%) or strong biofilm producers (5 out of 25; 26%; Table 2). A total of 35 MRSE isolates were obtained from the anterior nares of healthy individuals from the community. It was observed that 21 (60%) of these were able to produce biofilm. Of the 35 isolates, 3 (8%) were classified as weak, 9 (26%) as moderate and 9 (26%) as strong biofilm producers (Table 3). Finally, we tested the biofilm production in MRSE isolates obtained from anterior nares of a population associated with an HCS (total of 77 isolates), involving three categories: patients (10 isolates), their household contacts (29 isolates) and healthcare workers (38 isolates). It was observed that 57% (44 out of 77) of the total MRSE isolates obtained from the HCS produced biofilms on polystyrene surfaces. Among these 77 isolates, 12 (16%) were classified as weak, 7 (9%) as moderate and 25 (32%) as strong biofilm producers (Table 4). No statistical difference among these isolates was found for biofilm production when these three categories were compared. The percentages of biofilm production were 50% (5 out of 10) for MRSE isolates obtained from patients, 61% (23 out of 38) from healthcare workers and 55% (16 out of 29) from household contacts. Also, when the incidence of biofilm production among MRSE isolates from the three populations studied (HUAP, community and HCS; Figure 1) was compared, it was observed that there was no statistically significant difference between isolates from the healthy individuals from the community (60%) and patients, household contacts and healthcare workers from the HCS (57%). However, there was a significant statistical difference (P = 0.0107) for biofilm production between isolates obtained from infected patients from HUAP (76%) and nose isolates collected from the other two populations studied (community; 60% and HCS; 57%).
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Antimicrobial resistance
Among the total of 137 isolates studied, no resistance was detected to linezolid, vancomycin or teicoplanin. For many of the drugs tested, resistance was significantly higher among isolates obtained from patients from HUAP and from patients, household contacts and healthcare workers from the HCS than in MRSE isolates collected from healthy individuals from the community (P < 0.0001). For this reason, only for the analysis of antimicrobial resistance, MRSE isolates from patients from HUAP (25 isolates) and from patients, household contacts and healthcare workers from the HCS (77 isolates) were all grouped into the category of healthcare-associated isolates (total: 102 isolates). Resistance percentages of 48% (49 out of 102) for trimethoprim/sulphamethoxazole and 64% (65 out of 102) for clindamycin were obtained for the healthcare-associated isolates. On the other hand, a low value of 9% (3 out of 35) and no resistance (0 out of 35) were observed for healthy individuals from the community for these antimicrobials, respectively. In addition, resistance to ciprofloxacin (54%; 55 out of 102), chloramphenicol (45%; 46 out of 102) and rifampicin (36%; 37 out of 102) were higher among healthcare-associated isolates than among isolates from healthy individuals from the community (ciprofloxacin, 11%; 4 out of 35; no resistance for chloramphenicol and rifampicin). In contrast, the percentage of tetracycline resistance was higher among isolates collected from healthy individuals from the community (89%; 31 out of 35) than among healthcare-associated isolates (24%; 24 out of 102). There was no significant difference in the percentages of resistance to erythromycin [75% (76 out of 102) and 54% (19 out of 35), for healthcare-associated and community isolates, respectively], gentamicin [57% (58 out of 102) and 63% (22 out of 35)] and mupirocin [5% (5 out of 102) and no resistance] (Tables 2
–4).
Association of biofilm production with antimicrobial multiresistance
When drug multiresistance to three or more antimicrobials, other than ß-lactams, was correlated with biofilm formation, it was found that biofilm-producing MRSE isolates from healthy individuals from the community had a higher incidence of multiresistance (71%; 15 out of 21) than biofilm non-producers from the same population (0 out of 14; P < 0.0001; Figure 2). In addition, the six most susceptible isolates among the biofilm producers were weak or moderate producers. Moreover, all strong biofilm producers displayed antimicrobial multiresistance (Table 3). Similarly, in the group of household contacts from the HCS an increased incidence of multiresistance was noted (P < 0.0442) among biofilm-producers (87%; 14 out of 16) when compared with the biofilm non-producers (54%; 7 out of 13; Figure 2). However, there was no statistical difference for multiresistance between biofilm producers and biofilm non-producers among isolates from patients from HUAP and from patients and healthcare workers from the HCS (Tables 2 and 3).
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Detection of icaAD, atlE and aap genes
It was observed that the majority of the 84 biofilm-producing MRSE isolates detected carried the ica operon and atlE and aap genes (Tables 2–4 and Figure 3a). Thus, 83 (99%) of the isolates harboured the genes icaAD, 72 (86%) atlE and 69 (82%) aap. Only in one biofilm-producing MRSE isolate was none of these genes detected. This isolate (73/04D) was obtained from a healthy individual from the community and was classified as a moderate biofilm producer (Table 3). Since ica has been considered to be critical for the production of biofilm, the total DNA from this isolate was prepared and hybridized using a specific icaAD DNA probe. The result of this experiment confirmed the data obtained using PCR that this isolate lacked the genes icaAD (Figure 3b). It was also observed that a biofilm-non-producer MRSE isolate (CM133/03) from HUAP carried all the three genes (Table 2). In addition, two other isolates (HC328 and HC329) from the HCS population that harboured icaAD did not produce biofilms on polystyrene surfaces. The presence of icaAD in these isolates was confirmed using hybridization experiments (Figure 3b).
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DNA (positive control), isolate CM 133/03 (icaAD+, biofilm non-producer), HC 329 (icaAD+, biofilm non-producer), HC 328 (icaAD+, biofilm non-producer), 73/04 D (icaAD–, moderate biofilm producer).It is important to remark that all MRSE isolates displaying the strong biofilm-producer phenotype carried all the three genes (100%). Among the moderate biofilm producers, 62% (13 out of 21) harboured all the genes tested. Finally, only 58% of the weak producers (14 out of 24) carried the three genes. The statistical analysis showed that there was a significant association (P < 0.0001) between the detection of these genes and the biofilm expression profiles, indicating a tendency of the isolates that display the strong biofilm-producer phenotype to harbour the ica operon and atlE and aap concomitantly (Figure 4).
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In the work presented in this article biofilm production was studied in different populations of MRSE isolates obtained from patients from a university hospital (HUAP); from healthy individuals from the community; and from patients, household contacts and healthcare workers from an HCS. It was observed that the percentage of biofilm-producing MRSE isolates was significantly higher among hospital isolates (P = 0.0107). De Silva et al.31 observed that the amount of biofilm produced by S. epidermidis was significantly higher among isolates from either blood or skin of neonates with bacteraemia than among isolates from the skin of well-controlled neonates. In addition, Klingenberg et al. in their study including 150 cases of neonatal bacteraemia associated with S. epidermidis and other species of coagulase-negative staphylococci (CoNS) observed that the percentage of biofilm production was higher among S. epidermidis than in other CoNS isolates.22 All these data support the idea that biofilm production is an important virulence factor for the pathogenicity of S. epidermidis nosocomial isolates and would explain the high prevalence of this Staphylococcus species in medical-device-associated infections.5–10 Although the results obtained here revealed that biofilm formation was higher among hospital isolates than among nose isolates of non-infected individuals, it was also found that
60% of the commensal MRSE isolates obtained from the nares were able to produce biofilm. Similar results have been reported by other researchers, who observed a significant number of isolates capable of producing biofilm among S. epidermidis obtained from colonization cases.32 Thus, these data indicate that despite their role of biofilm production in S. epidermidis infections, both biofilm and the genes associated with this phenotype should not be used as markers for clinical significance, as suggested previously.20,21,33 Recently, other researchers also detected the presence of the ica operon in the great majority of biofilm-producing clinical isolates of S. epidermidis. Similar to the results obtained in this study, they showed that the detection of ica could not distinguish clinical isolates from commensal isolates.17,34 In the study presented in this article it was observed that the level of antimicrobial resistance was higher among healthcare-associated MRSE isolates than among community isolates. Similar results were also obtained previously.1,2 In addition, it was demonstrated that multiresistance was associated with biofilm production in the isolates obtained from healthy individuals from the community and from household contacts from the HCS (P < 0.0001). The association of biofilm production and multiresistance has been described previously by others.17,22,23 Thus, in addition to promoting bacterial adhesion to the surface of medical devices, biofilm production seems to have the potential to increase bacterial virulence by facilitating the genetic exchange of antimicrobial resistance genes. However, in this study, the association between biofilm production and multiresistance could not be observed among S. epidermidis isolates obtained from patients from HUAP and from patients and healthcare workers from the HCS. In these isolates, increased rates of resistance were detected for most of the drugs analysed, except for tetracycline, for which the percentage (89%) was higher among isolates from healthy individuals from the community. The great majority of HCS patients had hospital admissions just before their entry into the HCS. Moreover, most of the healthcare staff worked for other hospitals in addition to the HCS. Thus, these conflicting results could be explained by the enormous selective pressure exerted by the widespread use of antimicrobial drugs in hospitals. Similar results were observed by Kotilainen et al.,24 who found no difference in antimicrobial resistance between biofilm-producer and non-producer S. epidermidis isolates obtained from bloodstream infections.24
In addition to PIA, proteins have also been associated with biofilm formation by S. epidermidis, including Aap and AtlE proteins.11,19 The results presented in this article showed that the great majority of biofilm-producing MRSE isolates harboured the ica operon and the genes aap and atlE. With a few exceptions (4 out of 127) the presence of these genes was not detected among biofilm non-producers. These observations seem to reflect the importance of these genes for biofilm production in S. epidermidis. Many studies using S. epidermidis insertional mutants displaying an inactivation of the ica operon concluded that the functioning of this operon is essential for the biofilm formation and virulence of S. epidermidis.17,35–38 However, in the study presented here we found one ica-independent moderate biofilm producer (73/04 D) among these MRSE isolates. Similarly, the genes aap and atlE could not be detected in this isolate with the PCR primers used. This result indicates that other mechanisms of biofilm production may coexist in S. epidermidis. In fact, Fitzpatrick et al.39 in their study of a closely related bacterial species, S. aureus, reported that the deletion of the ica locus in three methicillin-resistant S. aureus clinical isolates did not result in a biofilm-negative phenotype and all three isolates displayed glucose-induced, ica-independent biofilm production. Recently, Tormo et al.40 reported that Bap orthologues from CoNS induce an alternative mechanism of biofilm formation that is independent of the PIA exopolysaccharide. Those authors demonstrated that all coagulase-negative staphylococcal isolates harbouring bap were strong biofilm-producers despite the fact they did not contain the icaADBC operon. Rohde et al.41 found that biofilm production in the clinically significant S. epidermidis 5179 depended on the expression of a truncated isoform of Aap. In addition, they observed that for the aap-negative S. epidermidis 1585, the expression of the truncated form of Aap mediates intercellular adhesion and biofilm production in a PIA-independent manner.
In addition, the data obtained here suggest that the simultaneous presence of ica, aap and atlE has an important role in the strong biofilm-producer phenotype (P < 0.0001). This finding is consistent with a role for aap and atlE in biofilm formation as suggested by previous studies using laboratory mutants.14,15 It was shown by others that the induction of stationary phase cells of S. epidermidis using glucose led to immediate PIA synthesis due to abundant amounts of functionally active ica mRNA.42 In spite of this, the isolate CM133/03, which harboured ica, aap and atlE genes, was not able to produce biofilm under these conditions. However, it was recently demonstrated that the rbf gene (encoding a putative transcriptional regulator) is involved in the expression of the multicellular aggregation step of S. aureus biofilm production in response to glucose and salt.43 Moreover, recent studies have shown that global regulators such as the Agr quorum sensing system,37 the transcriptional regulator SarA44 and the alternative sigma factor SigB45 also have important functions in the regulation of biofilm expression. Thus, a number of environmental conditions and regulatory systems can also influence the expression of staphylococcal biofilms, reflecting the magnitude of the complexity associated with the biofilm structure and its expression.
In conclusion, commensal MRSE isolates are also well equipped to produce biofilms and consequently biofilm should not be used as a marker for clinical significance. In addition, an association of the concomitant presence of ica, aap and atlE genes with the strong biofilm-producer phenotype was observed. The biofilm environment has been reported to promote the dissemination of antimicrobial resistance genes, probably by facilitating cell–cell contact and consequently increasing genetic transfer. The data observed in the present study corroborate these findings. Thus, although this hypothesis was not tested in this study, it is reasonable to suppose that the biofilm environment may potentially accelerate bacterial evolution towards genetic exchanges not only of resistance genes but also of other genetic elements related with bacterial adaptability and virulence.
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None to declare.
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Acknowledgements |
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This work was supported in part by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and PRONEX. We thank Sílvio José de Melo Júnior and Luiz Sérgio Keim for the isolates from Hospital Universitário Antônio Pedro and for the analysis of patient medical histories.
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References |
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