Abstract

Staphylococcus epidermidis, a member of the coagulase-negative staphylococci (CoNS) group, is the main bacterium found on human skin and causative agent of medical device-associated infections. The contamination of prostheses and intravenous devices with this pathogen is related to the production of a virulence factor, which represents an important pathogenic mechanism of implant infections. This substance, known as slime or biofilm, permits microorganisms to adhere to different materials (Costerton et al. 1995Costerton JW, Lewandowski Z, Cadwell DE, Korber DR, Lappin-Scott HM 1995. Microbial biofilms. Ann Rev Microbiol 49: 711-745.).

Biofilm formation is a process that involves microbial adhesion to and colonisation of a surface, cell proliferation and accumulation in multilayers, maturation and, finally, biofilm detachment and the release of cells (Houston et al. 2011Houston P, Rowe SE, Pozzi C, Waters EM, O’Gara JP 2011. Essential role for the major autolysin in the fibronectin-binding protein-mediated Staphylococcus aureus biofilm phenotype. Infect Immun 79: 1153-1165.). In addition to non-specific interactions such as electrostatic and hydrophobic interactions, specific adhesins, including two staphylococcal surface proteins (SSP-1 and SSP-2), are involved in the initial attachment to the polymer surface. When a certain material is implanted into an individual, the components of body fluids, such as serum proteins and platelets, start to cover the catheter or implant, modifying its surface properties and facilitating bacterial adhesion (Patti et al. 1994Patti JM, Allen BL, McGavin MJ, Hook M 1994. MSCRAMM-mediated adherence of microorganisms to host tissues. Ann Rev Microbiol 48: 585-617., von Eiff et al. 2002Von Eiff C, Peters G, Heilmann C 2002. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis 2: 677-685.). S. epidermidis and Staphylococcus aureus express dozens of proteins on their surfaces. These proteins, called microbial surface components recognising adhesive matrix molecules, specifically bind to extracellular matrix proteins of the host, such as fibrinogen, collagen, fibronectin and vitronectin (Patti et al. 1994Patti JM, Allen BL, McGavin MJ, Hook M 1994. MSCRAMM-mediated adherence of microorganisms to host tissues. Ann Rev Microbiol 48: 585-617., von Eiff et al. 2002Von Eiff C, Peters G, Heilmann C 2002. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis 2: 677-685.).

One of the main steps of biofilm formation is the production of polysaccharide intercellular adhesin (PIA), which is responsible for intercellular adhesion and the accumulation of cells in multilayers. The production of PIA is mediated by the products of the chromosomal intercellular adhesion genes (ica), which are organised into an operon that contains icaADBC genes, responsible for biosynthesis, and the icaR gene, which exerts a regulatory function (Otto 2008Otto M 2008. Staphylococcal biofilms. Curr Top Microbiol Immunol 322: 207-228.). PIA is synthesised from UDP-N-acetylglucosamine by N-acetylglucosamine transferase, an enzyme encoded by icaA and icaD genes, particularly icaA. The simultaneous expression of icaA and icaD promotes a significant increase in N-acetylglucosamine transferase (McCann et al. 2008McCann MT, Gilmore BF, Gorman SP 2008. Staphylococcus epidermidis device-related infections: pathogenesis and clinical management. J Pharm Pharmacol 60: 1551-1571., Otto 2008Otto M 2008. Staphylococcal biofilms. Curr Top Microbiol Immunol 322: 207-228.). Another possibility is that the protein IcaA (Mack et al. 1999Mack D, Riedewald J, Rohde H, Magnus T, Feucht HH, Elsner HA, Laufs R, Rupp ME 1999. Essential functional role of the polysaccharide intercellular adhesin of Staphylococcus epidermidis in hemagglutination. Infect Immun 67: 1004-1008.) requires IcaD to assume an active conformation. The icaC gene, when expressed concomitantly with icaA and icaD, induces the synthesis of longer oligomers. After export, PIA is deacetylated by the protein IcaB, which introduces positive charges that are crucial for the superficial localisation and biological function of PIA (Otto 2008Otto M 2008. Staphylococcal biofilms. Curr Top Microbiol Immunol 322: 207-228.).

In addition to PIA, adhesive proteins such as accumulation-associated protein and biofilm-associated protein (Bhp in S. epidermidis and Bap in S. aureus) may contribute to biofilm formation (Rupp et al. 2001Rupp ME, Fey PD, Heilmann C, Gotz F 2001. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter associated infection in a rat model. J Infect Dis 183: 1038-1042., Von Eiff et al. 2002Von Eiff C, Peters G, Heilmann C 2002. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis 2: 677-685.), even in the absence of the icaADCB operon. However, catheter-related and other nosocomial infections caused by biofilm-producing S. epidermidis are related to the presence of the ica operon, which is the main factor responsible for biofilm formation in this species (Cafiso et al. 2004Cafiso V, Bertuccio T, Santagati M, Campanile F, Amicosante G, Perilli MG, Selan L, Artini M, Nicoletti G, Stefanii S 2004. Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin Microbiol Infect 10: 1081-1088.).

The bacteria present inside a biofilm are protected against the action of the host immune system and antimicrobial drugs, thus permitting their survival (Mah & O’Toole 2001Mah TF, O’Toole GA 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9: 34-39., Donlan & Costerton 2002)Donlan RM, Costerton JW 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15: 167-193.. Biofilm-associated bacteria are usually less susceptible to antibiotics than planktonic bacteria; this can be explained by different mechanisms, such as the binding of antibiotics to biofilm components, reduced penetration of the antibiotic, slower growth of the microorganisms in the biofilm, high bacterial density and altered gene expression in the bacteria present in the biofilm (Stewart & Costerton 2001Stewart PS, Costerton JW 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358: 135-138., Singh et al. 2010)Singh R, Ray P, Das A, Sharma M 2010. Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms. J Antimicrob Chemother 65: 1955-1958..

Oxacillin, one of the antibiotics most commonly used in Brazil for the treatment of staphylococcal infections, is no longer effective because of the high prevalence of resistant strains. Resistance to methicillin is generally conferred by the mecA gene, which produces a penicillin-binding protein (PBP2a or PBP 2’) with reduced affinity for beta-lactam antibiotics when compared to other PBPs (Chambers et al. 1985Chambers HF, Hartman BJ, Tomasz A 1985. Increased amounts of a novel penicillin-binding protein in a strain of methicillin-resistant Staphylococcus aureus exposed to nafcillin. J Clin Invest 76: 325-331.).

The mecA gene is carried by a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec). Eleven different types of SCCmec have been identified in S. aureus (I-XI) (sccmec.org); however, types VI, VII, IX-XI have not yet been described in CoNS (Zong et al. 2011Zong Z, Peng C, Lu X 2011. Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. Plos ONE 6: e20191., Vitali et al. 2014Vitali LC, Petrelli D, Lamikanra A, Prenna M, Akinkunmi EO 2014. Diversity of antibiotic resistance genes and staphylococcal cassette chromosome mec elements in faecal isolates of coagulase-negative staphylococci from Nigeria. BMC Microbiology 14: 106.). SCCmec elements are more diverse in methicillin-resistant-CoNS (MR-CoNS) and new variants of the ccr genes continue to be identified, which cannot be typed with the currently available schemes. Thus, classification schemes of SCCmec in MR-CoNS are needed (Zong et al. 2011Zong Z, Peng C, Lu X 2011. Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. Plos ONE 6: e20191.). SCCmec types III-V are prevalent in MR-CoNS (Zong et al. 2011Zong Z, Peng C, Lu X 2011. Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. Plos ONE 6: e20191.). Type IV is the smallest, which reduces the cost of transfer between strains and selectively favours this type (Ito et al. 2001Ito T, Katayama Y, Asada K, Mori N, Tsutsumimoto K, Tiensasitorn C, Hiramatsu K 2001. Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 45: 1323-1336.).

The high prevalence of methicillin resistance has led to the use of toxic antibiotics, such as the glycopeptide vancomycin for the treatment of Gram-positive infections. However, intermediate resistance to this drug was described in Staphylococcus haemolyticus in the 1980s (Schwalbe et al. 1987Schwalbe RS, Stapleton JT, Gilligan PH 1987. Emergence of vancomycin resistance in coagulase negative staphylococci. N Engl J Med 316: 927-931.) and two strains (1 each of S. epidermidis and S. haemolyticus) with intermediate vancomycin resistance were found in Brazil in 1996 (Del’Alamo et al. 1999Del’Alamo L, Cerada RF, Tosin I, Miranda EA, Sader HS 1999. Antimicrobial susceptibility of coagulase-negative staphylococci and characterization of isolates with reduced susceptibility to glycopeptides. Diagn Microbiol Infect Dis 34: 185-191.). This is a matter of concern because few other options are currently available for the treatment of staphylococcal infections.

In view of the importance of biofilm formation and antimicrobial resistance in infections caused by S. epidermidis, the objective of the present study was to characterise S. epidermidis strains isolated from blood cultures of patients hospitalised at a Brazilian teaching hospital with regard to their methicillin resistance, SCCmec type, reduced susceptibility to vancomycin, presence of the ica genes and biofilm formation.

SUBJECTS, MATERIALS AND METHODS

A total of 107 S. epidermidis strains isolated from blood cultures of patients hospitalised at a teaching hospital at Botucatu Medical School (FMB), São Paulo State University (UNESP), state of São Paulo, Brazil, between 2005-2010 were studied. The University Hospital of FMB provides tertiary care and possesses 385 beds, including 52 in intensive care units (ICUs) (30 adult, 15 neonatal and 7 paediatric ICU beds). The isolates are stored in the Culture Collection of the Department of Microbiology and Immunology, Botucatu Institute of Biosciences, UNESP, and were obtained from adult and paediatric patients of both genders hospitalised in different units of the hospital. Table I describes the characteristics of the patients and the year of S. epidermidis isolation. The criteria proposed by the Centers for Disease Control and Prevention/National Healthcare Safety Network (CDC/NHSN 2014CDC/NHSN - Centers for Disease Control and Prevention/National Healthcare Safety Network 2014. Surveillance Definitions for specific types of infections. Available from: cdc.gov/nhsn/pdfs/pscmanual/17pscnosinfdef_current.pdf.
cdc.gov/nhsn/pdfs/pscmanual/17pscnosinfd... ) were used to determine the inclusion of blood cultures.

Species of the genus Staphylococcus were isolated and identified as described by Baker (1984)Baker JS 1984. Comparison of various methods for differentiation of staphylococci and micrococci. J Clin Microbiol 9: 875-879. and Koneman et al. (1997)Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn Jr WC 1997. Color atlas and textbook of diagnostic microbiology, 5th ed., Lippincott Williams & Wilkins, Philadelphia, 1395 pp.. The simplified method proposed by Cunha et al. (2004)Cunha MLRS, Sinzato YK, Silveira LVA 2004. Comparison of methods for identification of coagulase-negative staphylococci. Mem Inst Oswaldo Cruz 99: 855-860. was used for strain identification. DNA was extracted from isolates identified as S. epidermidis using the Illustra^® kit (GE Healthcare). Amplification of the internal transcribed spacer region-polymerase chain reaction (PCR), as described by Barry et al. (1991)Barry T, Colleran G, Glennon M, Dunican LK, Gannon F 1991. The 16S/23S ribosomal spacer region as a target for DNA probes to identify eubacteria. PCR Methods Appl 1: 51-56. and Couto et al. (2001)Couto I, Pereira S, Miragaia M, Sanches IS, Lencastre H 2001. Identification of clinical staphylococcal isolates from humans by internal transcribed spacer PCR. J Clin Microbiol 39: 3099-3103., was used to confirm that the isolates belonged to the species S. epidermidis.

The disk diffusion test employing oxacillin (1 μg) and cefoxitin (30 μg) disks was used for the phenotypic detection of methicillin resistance according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI 2007CLSI - Clinical and Laboratory Standards Institute 2007. Performance standards for antimicrobial susceptibility testing. Approved Standard Document M100-S17. Available from: microbiolab-bg.com/CLSI.pdf.
microbiolab-bg.com/CLSI.pdf... , 2012CLSI - Clinical and Laboratory Standards Institute 2012. Performance standards for antimicrobial susceptibility testing. Approved Standard Document M100-S22. Available from: antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/M100S22E.pdf.
antimicrobianos.com.ar/ATB/wp-content/up... ). For genotypic analysis, PCR was used to detect the mecA gene (Murakami et al. 1991Murakami K, Minamide K, Wada K, Nakamura E, Teraoka H, Watanabe S 1991. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 29: 2240-2244.). International reference strains were included in all reactions as positive (S. aureus ATCC 33591) and negative (S. aureus ATCC 25923) controls, according to the CLSI (2012)CLSI - Clinical and Laboratory Standards Institute 2012. Performance standards for antimicrobial susceptibility testing. Approved Standard Document M100-S22. Available from: antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/M100S22E.pdf.
antimicrobianos.com.ar/ATB/wp-content/up... . The strains that were positive for the mecA gene were subjected to SCCmec typing by multiplex PCR, according to Machado et al. (2007)Machado ABMP, Reiter KC, Paiva RM, Barth AL 2007. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microb 56: 1328-1333..

The presence of the icaA, icaD, icaB and icaC genes was determined by PCR (Arciola et al. 2001Arciola CR, Baldassarri L, Montanaro L 2001. Presence of icaA and icaD genes and slime production of staphylococcal strains from catheter-associated infections. J Clin Microbiol 39: 2151-2156., 2005Arciola CR, Gamberini S, Campoccia D, Visai L, Speziale P, Baldassari L, Montanaro L 2005. A multiplex PCR method for the detection of all five individual genes of ica locus in Staphylococcus epidermidis. A survey on 400 clinical isolates from prosthesis-associated infections. J Biomed Mater Res A 75: 408-413.) and the results were validated using international reference strains as positive (S. epidermidis ATCC 35983) and negative (S. epidermidis ATCC 12228) controls.

Biofilm production by the isolates that were positive for icaADBC was detected by the polystyrene plate method described by Christensen et al. (1985)Christensen GD, Simpson WA, Yonger JJ, Baddor LM, Barrett FF, Melton DM, Beachey EH 1985. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22: 996-1006. and modified by Oliveira and Cunha (2010). All isolates were screened for reduced susceptibility to vancomycin by growth on agar containing 4 and 6 μg mL^-1 of the antibiotic, as described by Hiramatsu (2001Hiramatsu K 2001. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis 1: 147-155. ) and CLSI (2012)CLSI - Clinical and Laboratory Standards Institute 2012. Performance standards for antimicrobial susceptibility testing. Approved Standard Document M100-S22. Available from: antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/M100S22E.pdf.
antimicrobianos.com.ar/ATB/wp-content/up... , but using an inoculum size of 2.0 McFarland standards. International reference strains (Enterococcus faecalis ATCC 29212 and ATCC 51299) were used as negative and positive controls, respectively. The presence of the vanA and vanB genes was determined by PCR (Clark et al. 1993Clark NC, Cooksey RC, Hill BC, Swenson JM, Tenover FC 1993. Characterization of glycopeptide-resistant enterococci from US hospitals. Antimicrob Agents Chemother 37: 2311-2317.) and the results were validated using international reference strains as positive (E. faecalis ATCC 51299) and negative (E. faecalis ATCC 29212) controls.

Vancomycin minimum inhibitory concentrations (MICs) were determined by a standardised broth microdilution method according to CLSI recommendations (CLSI 2012) and using panels prepared in-house. The following vancomycin concentrations were prepared with cation-adjusted Mueller-Hinton broth: 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 μg mL^-1. After inoculation, each well contained approximately 5 x 10⁵ colony-forming unit mL^-1. The MIC was defined as the concentration that completely inhibited bacterial growth after 24 h of incubation at 35ºC. However, for comparison, the plates were also analysed after 48 h. The following international reference strains were used to validate the results: E. faecalis ATCC 51299, E. faecalis ATCC 29212 and S. aureus ATCC 29213.

The results were compared by the chi-squared test, adopting a level of significance of < 0.05 (Curi 1997Curi PR 1997. Metodologia e análise da pesquisa em ciências biológicas, Tipomic, Botucatu, 263 pp.).

RESULTS

Seventy-nine (73.8%) of the 107 S. epidermidis isolates studied were resistant to methicillin based on the disk diffusion method using cefoxitin or oxacillin disks; 87 (81.3%) of the isolates were positive for the mecA gene. The sensitivity and specificity of the two phenotypic methods were 87.3% and 85%, respectively.

The mecA gene-positive isolates were subjected to SCCmec typing. Twenty-one (24.1%) of the 87 isolates were classified as SCCmec type I, one (1.1%) as type II, 42 (48.2%) as type III and 18 (20.7%) as type IV; five (5.7%) could not be typed by this technique. A decrease in strains carrying SCCmec type III was observed over the period from 2005-2007 (56.8%) and from 2008-2010 (39.5%), whereas the prevalence of type IV increased almost threefold (from 11.3 to 30.3%).

Vancomycin susceptibility testing showed that 51 (47.7%) isolates were able to grow on agar plates containing 4 μg mL^-1 of the antibiotic, whereas three (2.8%) isolates were able to grow on agar containing 6 μg mL^-1. The colonies were confirmed to be CoNS by Gram staining as well as catalase and coagulase tests to rule out the possibility of contamination. DNA was extracted from the colonies to determine the presence of the vanA and vanB genes and all of them were negative for these genes.

The vancomycin MICs obtained for subpopulations that grew on vancomycin screen agar and for the original populations are shown in Table II. The MIC was ≥ 4 μg mL^-1 in 64.7% of the isolates that grew on agar plates containing 4 μg mL^-1 vancomycin, with an MIC of 8 μg mL^-1 in 15.7% of these isolates and an MIC of 16 μg mL^-1 in one isolate (2%). MICs of 8 and 4 μg mL^-1 were observed in two (66.6%) and one of the three strains that grew on agar plates with 6 μg mL^-1 vancomycin, respectively. A comparison of MICs between the original isolates and the subpopulations that grew on vancomycin screen agar showed an increase in MIC in 75.9% of the isolates after 24 h of incubation. A two, four or eight-fold increase was observed in 96.3% of the strains after 48 h.

Regarding the presence of ica genes, at least one ica gene was detected in 96 (89.7%) of the isolates. Forty-one (38.3%) S. epidermidis isolates carried the complete ica operon, 43 (40%) carried the icaA+icaD+icaC genes, 47 (43.9%) concomitantly carried the icaA and icaD genes and 11 (10.9%) were negative for all genes of the operon. All icaA gene-positive isolates were positive for icaD (Table III). Twenty-four (58.5%) of the isolates carrying the complete ica operon produced a biofilm; of these, 15 (36.6%) were classified as weakly adherent and nine (22%) as strongly adherent.

Eight (33.3%) of the isolates that produced a biofilm and carried the icaADBC operon exhibited vancomycin MICs ≥ 8 μg mL^-1, whereas this level of resistance was observed in only nine (8.4%) of all isolates studied. The only isolate that exhibited a vancomycin MIC equal to 16 μg mL^-1 produced a strongly adherent biofilm.

Comparisons of the isolates that exhibited an increase in the vancomycin MIC after 24 h of incubation (n = 39; 36.4%) with those that did not grow (n = 55; 51.4%) and those that did grow, but maintained the same MIC (n = 13; 12.1%), revealed that the increase in vancomycin MIC was positively associated with methicillin resistance using oxacillin or cefoxitin disks (p = 0.02) and the presence of SCCmec type III (p = 0.0003).

An association was observed between the presence of the mecA gene and the presence of the icaA+icaD genes (p = 0.001): most isolates carrying the mecA gene were also positive for the icaA+icaD+icaC genes (p = 0.002) and also for the complete icaADBC operon (p = 0.004). In addition, the presence of icaA and icaD was associated with SCCmec type III (p = 0.030) (Table III). Furthermore, the presence of the complete ica operon and biofilm production were significantly associated with the presence of the mecA gene (100%; p = 0.006) and with growth on vancomycin screen agar (66.7%; p = 0.05) when compared to isolates that did not carry the complete operon or that did carry the operon, but did not produce a biofilm.

DISCUSSION

Formerly considered to be an innocuous commensal bacterium of human skin, S. epidermidis is now recognised as an important opportunistic pathogen. This microorganism is one of the most common causative agents of medical device-related infections (Sievert et al. 2013Sievert DM, Ricks P, Edwards JR, Schneider A, Patel J, Srinivasan A, Kallen A, Limbago B, Fridkin S, for the National Healthcare Safety Network (NHSN) Team and Participating NHSN Facilities 2013. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infect Control Hosp Epidemiol 34: 1-14.). We isolated 107 S. epidermidis strains from blood cultures of patients seen at a Brazilian teaching hospital and ana- lysed antimicrobial resistance patterns, the presence of ica operon genes and biofilm formation. The results showed a high prevalence of methicillin resistance as detected by both phenotypic and genotypic methods (73.8% and 81.3%, respectively). This finding was expected because the prevalence of methicillin resistance is high (70-80%) among CoNS in Latin America (Diekema et al. 2001Diekema DJ, Pfaller MA, Schmitz FJ, Smayevsky J, Bell J, Jones RN, Beach M, SENTRY Participants Group 2001. Survey of infections due to Staphylococcus species: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, Latin America, Europe and the Western Pacific region for the SENTRY Antimicrobial Surveillance Program, 1997-1999. Clin Infect Dis 32: 114-132.).

The prevalence of SCCmec types III (48.2%) and IV (20.7%) was high, which is a finding that was also reported by Wisplinghoff et al. (2003)Wisplinghoff H, Rosato AE, Enright MC, Noto M, Craig W, Archer GL 2003. Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates. Antimicrob Agents Chemother 47: 3574-3579.. SCCmec type IV is smaller and the metabolic cost of its transfer is thus lower; as a consequence, its prevalence is expected to increase over time. Indeed, in the present study, the proportion of SCCmec type IV increased three-fold over a period of six years. Conversely, the prevalence of type III decreased during the same period, although its prevalence continues to be high. This high proportion of SCCmec type III might be related to the selection of this type in the hospital environment due to the multidrug resistance of these strains. These facts should be considered in infection control policies.

SCCmec type II was detected in only one isolate. A similar frequency was reported by Machado et al. (2007)Machado ABMP, Reiter KC, Paiva RM, Barth AL 2007. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microb 56: 1328-1333., suggesting that MR S. epidermidis (MRSE) carrying this SCCmec type is not prevalent in Brazil. SCCmec type I, a hospital-associated type, was frequent (24.1%); this finding stands in contrast to previous studies in which type I was found to be rare (Ibrahem et al. 2009Ibrahem S, Salmenlinna S, Virolainen A, Kerttula AM, Lyytikäinen O, Jägerroos H, Broas M, Vuopio-Varkila J 2009. Carriage of methicillin-resistant staphylococci and their SCCmec types in a long-term-care facility. J Clin Microbiol 47: 32-37., Barbier et al. 2010Barbier F, Lebeaux D, Hernandez D, Delannoy AS, Caro V, François P, Schrenzel J, Ruppé E, Gaillard K, Wolff M, Brisse S, Andremont A, Ruimy R 2010. High prevalence of the arginine catabolic mobile element in carriage isolates of methicillin-resistant Staphylococcus epidermidis. J Antimicrob Chemother 66: 29-36., Zong et al. 2011Zong Z, Peng C, Lu X 2011. Diversity of SCCmec elements in methicillin-resistant coagulase-negative staphylococci clinical isolates. Plos ONE 6: e20191.), but is similar to the findings reported in other Brazilian studies (Machado et al. 2007Machado ABMP, Reiter KC, Paiva RM, Barth AL 2007. Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil. J Med Microb 56: 1328-1333., Pereira & Cunha 2013Pereira VC, Cunha MLRS 2013. Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures. Mem Inst Oswaldo Cruz 108: 939-942., Ternes et al. 2013)Ternes YM, Lamaro-Cardoso J, André MCP, Pessoa VP, Vieira MAS, Minamisava R, Andrade AL, Kipnis A 2013. Molecular epidemiology of coagulase-negative Staphylococcus carriage in neonates admitted to an intensive care unit in Brazil. BMC Infect Dis 13: 572.. These findings might be related to the local epidemiology of MRSE, indicating a higher frequency of SCCmec type I in Brazilian hospitals. These data indicate the existence of a vast reservoir of SCCmec among S. epidermidis strains (Wisplinghoff et al. 2003)Wisplinghoff H, Rosato AE, Enright MC, Noto M, Craig W, Archer GL 2003. Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates. Antimicrob Agents Chemother 47: 3574-3579.. Although these similarities prevail, the distribution of SCCmec types depends on several factors, including geographic location and the use of antimicrobial agents.

The incidence of the ica genes was high in the present study, with 90.6% of the isolates being positive for at least one ica gene and 38.3% being positive for all genes. Biofilm production was detected by the phenotypic method in 58.5% of the isolates carrying the complete ica operon. Similarly, Oliveira and Cunha (2010) observed that 56.6% of strongly adherent CoNS carried the icaA+icaC+icaD genes. The regulation of the ica operon is complex and the expression of the ica genes is variable: it can be activated or deactivated according to in vivo conditions. Some events, such as the addition of the insertion sequence IS256, appear to be associated with this phenomenon (Ziebuhr et al. 1999Ziebuhr W, Krimmer V, Rachid S, Lössner I, Götz F, Hacker J 1999. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol Microbiol 32: 345-356.).

In the present study, all isolates that were positive for the icaA gene were also positive for icaD, in agreement with Arciola et al. (2001)Arciola CR, Baldassarri L, Montanaro L 2001. Presence of icaA and icaD genes and slime production of staphylococcal strains from catheter-associated infections. J Clin Microbiol 39: 2151-2156. and Gad et al. (2009)Gad GF, El-Feky MA, El-Rehewy MS, Hassan MA, Abolella H, El-Baky RM 2009. Detection of icaA, icaD genes and biofilm production by Staphylococcus aureus and Staphylococcus epidermidis isolated from urinary tract catheterized patients. J Infect Dev Ctries 3: 342-351.. Cafiso et al. (2004)Cafiso V, Bertuccio T, Santagati M, Campanile F, Amicosante G, Perilli MG, Selan L, Artini M, Nicoletti G, Stefanii S 2004. Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin Microbiol Infect 10: 1081-1088. showed that the icaD gene was always expressed in S. epidermidis, but that phenotypic biofilm production only occurred when icaA was expressed simultaneously. This relationship might be explained by the selective pressure exerted by biofilm production because, when expressed, the icaD gene alone does not induce transferase activity and icaA induces little activity; in contrast, the combined expression of icaA and icaD produces large amounts of PIA (Gerke et al. 1998Gerke C, Kraft A, Sussmuth R, Schweitzer O, Götz F 1998. Characterization of the N- acetylglucosaminyltransferase activity involved in the biosynthesis of the Staphylococcus epidermidis polysaccharide intercellular adhesion. J Biol Chem 273: 1856-1893.). Although the icaA and icaD genes overlap and are co-transcribed, the fact that the isolates were more frequently positive for icaD than icaA, icaB and icaC in the present study and in previous studies (Zhou et al. 2013Zhou S, Chao X, Fei M, Dai Y, Liu B 2013. Analysis of S. epidermidis icaA and icaA genes by polymerase chain reaction and slime production: a case control study. BMC Infect Dis 13: 242.) may be related to the loss or mutation of one of these genes.

The mecA gene was found to be associated with the presence of the icaADBC operon and biofilm production (p < 0.05). In addition, the concomitant presence of icaA+icaD was associated with SCCmec type III. Zhou et al. (2013)Zhou S, Chao X, Fei M, Dai Y, Liu B 2013. Analysis of S. epidermidis icaA and icaA genes by polymerase chain reaction and slime production: a case control study. BMC Infect Dis 13: 242. also detected icaD more frequently than icaA and observed a significant association between icaD and the mecA gene in S. epidermidis. Furthermore, other studies have demonstrated that methicillin resistance is correlated with biofilm production and the presence of ica genes (Alcaráz et al. 2003Alcaráz LE, Satorres SE, Lucero RM, Puig de Centorbi ON 2003. Species identification, slime production and oxacillin susceptibility in coagulase-negative staphylococci isolated from nosocomial specimens. Braz J Microbiol 34: 45-51., Abassi et al. 2008Abassi MS, Bouchami O, Touati A, Achour W, Hassen AB 2008. Clonality and occurrence of genes encoding antibiotic resistance and biofilm in methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and bacteremia in neutropenic patients. Curr Microbiol 57: 442-448., Koksal et al. 2009)Koksal F, Yasar H, Samasti M 2009. Antibiotic resistance patterns of coagulase-negative staphylococcus strains isolated from blood cultures of septicemic patients in Turkey. Microbiol Res 164: 404-410.. These associations might be a consequence of the intimate contact between biofilm bacteria, facilitating the horizontal transfer of genetic material such as antimicrobial resistance genes (Klingenberg et al. 2005)Klingenberg C, Aarag E, Ronnestad A, Sollid JE, Abrahamsen TG, Kjeldsen G, Flaegstad T 2005. Coagulase-negative staphylococcal sepsis in neonates: association between antibiotic resistance, biofilm formation and the host inflammatory response. Pediatr Infect Dis 24: 817-822., which would explain the horizontal transfer of mecA to ica gene-positive strains (Kozitskaya et al. 2004)Kozitskaya S, Cho SH, Dietrich K, Marre R, Naber K, Ziebuhr W 2004. The bacterial insertion sequence element IS256 occurs preferentially in nosocomial Staphylococcus epidermidis isolates: association with biofilm formation and resistance to aminoglycosides. Infect Immun 72: 1210-1215.. The present results suggest the presence of the icaADBC operon in conjunction with biofilm production and the presence of the mecA gene to be an important marker of nosocomial strains with the potential to cause infection. These markers are therefore useful for evaluating the clinical significance of CoNS.

The indiscriminate use of antibiotics has led to the selection of bacterial populations that contain cells with distinct levels of susceptibility to antimicrobial agents, including resistant cells. Intermediate and complete resistance to vancomycin in S. epidermidis has been reported (Palazzo et al. 2005Palazzo ICV, Araujo MLC, Darini ALC 2005. First report of vancomycin-resistant staphylococci isolated from healthy carriers in Brazil. J Clin Microbiol 43: 179-185.) and biofilm formation is known to significantly reduce the effects of this antibiotic on bacteria (Raad et al. 2007Raad I, Hanna H, Jiang Y, Dvorak T, Reitzel R, Chaiban G, Sherertz R, Hachem R 2007. Comparative activities of daptomycin, linezolid and tigecycline against catheter-related methicillin resistant Staphylococcus bacteremic isolates embedded in biofilm. Antimicrob Agents Chemother 51: 1656-1660.). In the present study, 47.7% and 2.8% of the S. epidermidis isolates grew colonies on screen agar plates containing 4 and 6 μg mL^-1 vancomycin, respectively. One-third of the isolates that were biofilm producers also exhibited vancomycin MICs ≥ 8 μg mL^-1 after growing on vancomycin screen agar, characteristics that can lead to treatment failure and may be a precursor of glycopeptide resistance.

The term vancomycin heteroresistance refers to strains that contain a subpopulation of cells with different levels of susceptibility to this antibiotic, including resistant cells and cells with intermediate susceptibility (Raad et al. 2007Raad I, Hanna H, Jiang Y, Dvorak T, Reitzel R, Chaiban G, Sherertz R, Hachem R 2007. Comparative activities of daptomycin, linezolid and tigecycline against catheter-related methicillin resistant Staphylococcus bacteremic isolates embedded in biofilm. Antimicrob Agents Chemother 51: 1656-1660.). Our analysis of colonies grown on vancomycin screen agar showed an increase in MICs in 75.9% of the isolates, with an increase from 1 to ≥ 4 μg mL^-1 in 64.7% of the isolates. MIC increases were also observed in 100% of the colonies that grew on agar with 6 μg mL^-1 vancomycin. Using isolates that grew on screen agar with 4 μg mL^-1 vancomycin, Ma et al. (2011)Ma XX, Wang EH, Liu Y, Luo EJ 2011. Antibiotic susceptibility of coagulase-negative staphylococci (CoNS): emergence of teicoplanin non-susceptible CoNS strains with inducible resistance to vancomycin. J Med Microbiol 60: 1661-1668. reported slightly lower rates: 64.7% of strains showed increased MICs and 35.3% showed MICs ≥ 4 μg mL^-1. MICs ≥ 4 μg mL^-1 were observed in 85.7% of the subpopulations that grew on screen agar with 6 μg mL^-1 vancomycin. In our study, increased MICs were detected in 96.3% of the isolates after 48 h of incubation. This finding confirms the existence of heteroresistant isolates.

The existence of vancomycin heteroresistance in nosocomial S. epidermidis is an alarming finding. Furthermore, the observation of subpopulations with MICs > 4 μg mL^-1 (64.7%), as well as MICs of 8 μg mL^-1 (15.7%) and 16 μg mL^-1 (2%), classifying the isolates as intermediate resistant, indicates that infections caused by these microorganisms will be difficult to treat. Indeed, reports have shown higher mortality among patients infected with heteroresistant bacteria and strains with higher MICs compared to those infected with susceptible strains (Wong et al. 1999)Wong SSY, Ho PL, Woo PCY, Yuen KY 1999. Bacteremia caused by staphylococci with inducible vancomycin heteroresistance. Clin Infect Dis 29: 760-767.. However, therapeutic alternatives for infections caused by staphylococci with reduced vancomycin resistance have been approved by the US Food and Drug Administration and include linezolide, daptomycin, tigecycline and quinupristin/dalfopristin (Micek 2007)Micek ST 2007. Alternatives to vancomycin for the treatment of methicillin-resistant Staphylococcus aureus infections. Clin Infect Dis 45 (Suppl. 3): S184-S190.. Two next-generation cephalosporins, ceftobiprole and ceftaroline fosamil, are currently available for the treatment of patients carrying MR and vancomycin-intermediate staphylococci (Saravolatz et al. 2011Saravolatz LD, Stein GE, Johnson LB 2011. Ceftaroline: a novel cephalosporin with activity against methicillin-resistant Staphylococcus aureus. Clin Infect Dis 52: 1156-1163., Lovering et al. 2012)Lovering AL, Gretes MC, Safadi SS, Danel F, Castro L, Page MGP, Natalie CJ, Strynadka NCJ 2012. Structural insights into the anti-methicillin-resistant Staphylococcus aureus (MRSA) activity of ceftobiprole. J Biol Chem 287: 32096-32102..

In the present study, reduced susceptibility to vancomycin was significantly associated with methicillin resistance detected by phenotypic methods (p < 0.05). Other studies (Wong et al. 1999Wong SSY, Ho PL, Woo PCY, Yuen KY 1999. Bacteremia caused by staphylococci with inducible vancomycin heteroresistance. Clin Infect Dis 29: 760-767., Loomba et al. 2010)Loomba PS, Taneja J, Mishra B 2010. Methicillin and vancomycin resistant S. aureus in hospitalized patients. J Glob Infect Dis 2: 275-283. have shown that the isolation of MR staphylococci is significantly more common among patients with bacteraemia due to vancomycin-heteroresistant staphylococci. Although the mechanism underlying the association between methicillin resistance and reduced vancomycin susceptibility remains unclear, it might be related to the treatment of infections caused by MR bacteria with vancomycin, thereby selecting heterogeneous populations and strains with low susceptibility.

The proportion of strains with reduced susceptibility to vancomycin was higher among the isolates harbouring SCCmec type III. Differing results have been reported by Moise et al. (2007)Moise PA, Sakoulas G, Forrest A, Schenag JJ 2007. Vancomycin in vitro bactericidal activity and its relationship to efficacy in clearance of methicillin-resistant Staphylococcus aureus bacteremia. Antimicrob Agents Chemother 57: 2582-2586., who found that higher vancomycin MICs were significantly associated with SCCmec type II in S. aureus, whereas lower MICs were related to SCCmec type IV. The association between reduced vancomycin susceptibility and SCCmec type III observed in the present study may be due to the high prevalence of SCCmec type III in the hospital environment and the frequent use of vancomycin.

The presence of subpopulations with higher vancomycin MICs and intermediate resistance demonstrates the heterogeneity of S. epidermidis strains in terms of susceptibility to this antibiotic. Because the main genes responsible for vancomycin resistance (vanA and vanB) were not detected, this reduced susceptibility may be related to a thickening of the bacterial cell wall that prevents uptake of the antibiotic by the microorganism (Billot-Klein et al. 1996Billot-Klein D, Gutmann L, Bryant D, Bell D, Van Heijenoort J, Grewal J, Shlaes DM 1996. Peptidoglycan synthesis and structure in Staphylococcus haemolyticus expressing increasing levels of resistance to glycopeptides antibiotics. J Bacteriol 178: 4696-4703.). However, further studies are needed to elucidate how these events occur in staphylococci.

The present study demonstrated an association between the presence of the icaADBC operon, biofilm formation and antimicrobial resistance in S. epidermidis strains isolated from blood cultures. Furthermore, the detection of S. epidermidis subpopulations with intermediate vancomycin resistance associated with methicillin resistance highlights the role of this species as an important multidrug-resistant microorganism and the consequent need to implement measures for the control of antibiotic use.

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