Antimicrobial synergism against different lineages of methicillin-resistant Staphylococcus aureus carrying SCCmec IV

Authors

  • P.D.M. de Matos,

    1. Department of Medical Microbiology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Nova Friburgo, Rio de Janeiro, Brazil
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  • S. Sedaca,

    1. Department of Medical Microbiology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Nova Friburgo, Rio de Janeiro, Brazil
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  • D.C. Ferreira,

    1. Department of Medical Microbiology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Nova Friburgo, Rio de Janeiro, Brazil
    2. Department of Oral Medicine, School of Dentistry, Veiga de Almeida University, Rio de Janeiro, Brazil
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  • N.L. Iorio,

    1. Basic Science Department, Fluminense Federal University, Nova Friburgo, Rio de Janeiro, Brazil
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  • V.C.S. Toledo,

    1. Department of Medical Microbiology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Nova Friburgo, Rio de Janeiro, Brazil
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  • A.I.C. Freitas,

    1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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  • F.L. Coelho,

    1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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  • C. Sousa,

    1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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  • K.R.N. dos Santos,

    Corresponding author
    1. Department of Medical Microbiology, Institute of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Nova Friburgo, Rio de Janeiro, Brazil
    • Correspondence

      Kátia Regina N. dos Santos, Laboratório de Infecções Hospitalares, Departamento de Microbiologia Médica, Instituto de Microbiologia Prof. Paulo de Góes, CCS, Bloco I, UFRJ, Cidade Universitária, Rio de Janeiro, CEP: 21941-590, RJ, Brazil.

      E-mail: santoskrn@micro.ufrj.br

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  • M.O. Pereira

    1. Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal
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Abstract

Aim

To evaluate the synergistic activity of antimicrobial drugs against lineages of methicillin-resistant Staphylococcus aureus (MRSA) carrying SCCmec IV. The biofilm production and related genes were also detected.

Methods and Results

Forty two MRSA isolates were tested for biofilm production and related genes. Biofilm/biomass susceptibility to gentamicin (G), linezolid (L), rifampicin (R) and vancomycin (V) was determined for six isolates from three lineages prevalent in Rio de Janeiro hospitals in concentrations ranging from 0·25 to 64 μg ml−1. Biomass was evaluated by microtitre plate test and number of viable cells (CFU cm−2) and inspected by epifluorescence microscopy. All isolates presented the icaA and sasG genes, but only 38% were biofilm producers. There were 50 and 45% biomass reductions when concentrations ≥4 μg ml−1 of R or L and ≥16 μg ml−1 of G or V, respectively, were used. Synergism tests produced a 55% biomass reduction with math formula + math formula, math formula + math formula, math formula  + math formula, and math formula + math formula. Number of viable cells was reduced from 2 to 3 logs with math formula + math formula and math formula + math formula.

Conclusions

Synergisms involving R plus L and R plus V caused important reductions in biofilm/biomass and the number of viable cells. Drug combinations should be considered in the chemotherapies of MRSA-SCCmec IV infections.

Significance and Impact of the Study

Biofilms in MRSA infections restrict the clinical choice of antimicrobials. Thus, knowledge of the best options for monotherapy and drug synergisms could improve clinical results.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen (David and Daum 2010). MRSA isolates present a penicillin-binding protein, PBP2a, encoded by the mecA gene that is inserted in a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec) (Katayama et al. 2000). Among the twelve different SCCmec allotypes already described (IWG-SCC 2013), type IV, which is related to the community isolates, has been emerging in hospitals worldwide causing infections in patients with or without traditional risk factors for MRSA (Schuenck et al. 2009; Holzknecht et al. 2010; Lesosky et al. 2011; Caboclo et al. 2013; Velasco et al. 2012).

In Brazil, MRSA isolates related to the Brazilian endemic clone (BEC)/sequence type (ST) 239 are traditionally found in hospital-acquired infections (Santos et al. 1999). However, over the last decade, changes in hospital epidemiology have been observed, with the emergence of nosocomial infections caused by SCCmec IV isolates, including such predominant lineages as USA400/ST1 (MW2 clone), USA1100/ST30 (Ocean Pacific clone) and USA800/ST5 (paediatric clone) (Silva-Carvalho et al. 2009; Schuenck et al. 2012; Caboclo et al.2013). Our group showed the polyclonal emergency of nonmultiresistant MRSA-SCCmec IV isolates in health-care-associated infections causing deaths in patients in a hospital in Rio de Janeiro (Schuenck et al. 2009).

Biofilm formation by Staph. aureus isolates is common in medical devices such as catheters and prostheses, allowing the pathogen to resist host immune responses and antimicrobials (Donlan and Costerton 2002). However, some studies have shown that the majority of type IV isolates do not produce biofilms or are weak or moderate biofilm formers (Cha et al. 2011; Schuenck et al. 2012). Moreover, the action of antimicrobial agents either individually or in combination against MRSA-SCCmec IV isolates has not yet been analysed. Thus, this study aimed to evaluate the activity of antimicrobial agents alone and in various combinations against the biofilm/biomass produced by different lineages of MRSA-SCCmec IV isolates from hospitals in Rio de Janeiro. Biofilm production and related genes were also evaluated.

Materials and methods

Clinical isolates

Of a collection of 128 MRSA-SCCmec IV isolates previously characterized as to species (Bannerman and Peacock 2007), methicillin resistance (CLSI 2012) and SCCmec type (Milheiriço et al. 2007), 42 were selected for this study. This selection was based on the DNA polymorphism profiles determined by the pulsed-field gel electrophoresis (PFGE) (Vivoni et al. 2006) and multilocus sequence typing (MLST) methods (Enright et al. 2000). The lineages were classified according to McDougal et al. (2003) as USA800/ST5 (14 isolates), USA400/ST1 (9) and USA1100/ST30 (4); the remaining 15 isolates belonged to other STs and/or clonalities. These isolates were obtained between July 2004 and November 2008 from different clinical specimens from patients in eight hospitals in Rio de Janeiro city (Table 1).

Table 1. General characteristics and biofilm formation ability of 42 MRSA-SCCmec IV isolates, positive for icaA and sasG genes, isolated from hospitals in Rio de Janeiro
Sequence type by MLST (n)Clonality by PFGEClinical source (n)Biofilm production (n)
  1. MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; ND, not determined; strong biofilm producer (+++), moderate biofilm producer (++), weak biofilm producer (+) and nonbiofilm producer (−).

1 (9)USA400Prosthesis secretion (3), urine (3), nasal (1), ear secretion (1), bronchial alveolar lavage (1)+ (1), − (8)
5 (14)USA800Nasal (4), wound (4), prosthesis secretion (3), bronchial alveolar lavage (2), blood (1)++ (2), + (6), − (6)
30 (4)USA1100Wound (2), bone secretion (1), renal abscess (1)+ (2), − (2)
5 (2)NDNasal (2),++ (1), + (1)
1203 (2)NDCatheter tip (1), wound (1)− (2)
97 (3)NDPleural fluid (1), nasal (2)++ (1), − (2)
8 (1)USA 300Wound++
22 (1)EMRSA-15Tracheal secretion
45 (1)USA 600Nasal
72 (1)NDBlood
2102 (1)NDTracheal secretion
714 (1)NDWound+
30 (1)NDBronchial alveolar lavage
2112 (1)NDNasal

Biofilm/biomass formation assay

Biofilm/biomass formation was evaluated for all 42 MRSA-SCCmec IV isolates according to the microtitre plate test protocol modified from that described by Stepanovic et al. (2000). A bacterial suspension in Tryptic Soy Broth (TSB, Merck, Algés, Portugal) supplemented with 1% glucose and adjusted to a final concentration of c. 1 × 107 cells ml−1 was transferred to a microtitre plate (200 μl per well). The plates were incubated aerobically on a horizontal shaker at 120 rpm at 37°C. After 24 h, the content of each well was removed and the wells were washed twice with 200 μl of sterile water. The plates were air-dried for 20 min, and bacterial biomass adhering to the inner surfaces of each microtitre plate well was fixed with 200 μl of 98% metanol (Vaz Pereira, Portugal) per well during 15 min. Afterwards, the plates were emptied, left to dry for 20 min, and fixed biomass was stained for 5 min with 200 μl of crystal violet (CV) (Merck) per well. Excess stain was rinsed off by placing the plate under running tap water. After the plate was air-dried, the dye bound to the adherent cells was resuspended with 200 μl of 33% (v/v) glacial acetic acid (Merck) per well. The optical density (OD) of the obtained solution was measured at 570 nm using a microtitre plate reader (Tecan, Model Sunrise-basic Tecan, Grödig, Austria), and the ability of Staph. aureus isolates to produce biofilm/biomass was evaluated as OD570 values.

The Staphylococcus epidermidis strains, ATCC 35984 (strong biofilm producer) and ATCC12228 (nonbiofilm producer) were used as controls. All isolates were classified in terms of their ability to form biofilm based on absorbance values into the following categories: strong producer (+++, OD > 2·0), moderate producer (++, OD from 1·0 to 2·0), weak producer (+, OD from 0·5 to <1·0) and non producer (−, OD < 0·5) of biofilm.

PCR method to detect biofilm-related genes

PCR was performed for the 42 MRSA-SCCmec IV isolates to detect icaA (Mirani and Jamil 2011) and sasG (Roche et al. 2003) genes, which encode a cell surface protein related to biofilm and the polysaccharide intercellular adhesin (PIA), respectively. The primer sequences used were ICAAF – 5′- AAACTTGGTGCGGTTACAGG-3′ and ICAAR – 5′-TCTGGGCTTGACCATGTTG-3′ (icaA gene), and SASGF – 5′-GAGATAAGAAAGGACCGG and SASGR – TTAATTCTTTCTTCTACG-3′ (sasG gene).

Bacterial biomass susceptibility testing

Biomass susceptibility to gentamicin (G), linezolid (L), rifampicin (R) and vancomycin (V) (Sigma-Aldrich, St. Louis, MO) were determined for six isolates selected from three prevalent lineages: USA400/ST1 (isolates 633, weak biofilm producer, and 915, nonproducer), USA800/ST5 (isolates 1112, weak biofilm producer, and 1177, moderate biofilm producer) and USA1100/ST30 (isolates 943, weak biofilm producer, and 1314, nonproducer). After biomass formation for 24 h, the isolates were exposed to antimicrobials, either individually or in combination, with concentrations ranging from 0·25 to 64 μg ml−1. The concentrations used were based on the antimicrobial break points (CLSI 2012). After antibiotic treatment for 24 h, the biofilms were characterized in terms of their biomass through the microtitre plate test (above described) and the number of cultivable cells, by plate count agar (CFU cm−2). To determine the number of CFU, the biomass formed within the wells was removed by rapid sonication for 6 min and, subsequently, serially diluted. After plating the serial dilution on Tryptic Soy Agar (TSA, Merck), the plates were incubated at 37°C, in an aerobic incubator for 18 h prior to enumeration.

Statistical methods

Data were recorded as the mean standard deviation. Because of the small sample size and the abnormal distribution, the Kruskal–Wallis test was used for multiple comparison analysis. Statistical significance was set at < 0·05. Data were analysed using spss 17.0 for Windows (SPSS Inc., Chicago, IL).

Results

Biofilm formation assay and related genes

The biofilm production was considered positive only for 38% of MRSA-SCCmec IV isolates, being 8 USA800/ST5 isolates (six weak biofilm producers and two moderate), 1 USA400/ST1 isolate that was considered a weak biofilm producer, 2 USA1100/ST30 isolates that were detected as weak biofilm producers and five other isolates from other clonalities were two weak biofilm producers and three moderate (Table 1). The 42 MRSA isolates presented the expected amplification bands of 751 bp and 300 bp for icaA and sasG genes, respectively (data not shown).

Biomass susceptibility testing

To assess the activity of antimicrobials against biofilm/biomass, drugs to which the MRSA-SCCmec IV isolates presented susceptibility in disc diffusion tests (data not shown) and that are usually indicated for treatment of MRSA infections were selected (Colli et al. 2007). After exposure to concentrations equal to or >4 μg ml−1 of R or L, a biomass reduction of 50% was observed. For G or V, a reduction of about 45% was verified using concentrations ≥16 μg ml−1 for all six MRSA-SCCmec IV isolates evaluated, irrespective of the lineage (Fig. 1). Regarding the cell viability analysis, which was determined by log CFU cm−2, a reduction of up to 1 log for math formula or math formula or 2 log for math formula or math formula was observed for all isolates (Fig. 2).

Figure 1.

Biomass evaluated after the use of increasing concentrations of antimicrobial agents (0·25–64 μg ml) for the six isolates from three MRSA-SCCmec IV lineages in relation to the positive control (PC) without antimicrobial exposure. Filled squares, triangles and circles represent 633 (USA400/ST1), 1177 (USA800/ST5) and 943 (USA1100/ST30) biofilm producer isolates; open squares, circles and triangles represent 915 (US400/ST1) and 1314 (USA1100/ST30) nonbiofilm producer isolates, and 1112 (USA800/ST5) weak producer isolate. (image_n/jam12472-gra-0001.png) 633 (USA400/ST1); (image_n/jam12472-gra-0002.png) 915 (US400/ST1); (image_n/jam12472-gra-0003.png) 1177 (USA800/ST5); (image_n/jam12472-gra-0004.png) 1112 (USA800/ST5); (image_n/jam12472-gra-0005.png) 943 (USA1100/ST30) and (image_n/jam12472-gra-0006.png)1314 (USA1100/ST30).

Figure 2.

Cell viability (log CFU cm−2) evaluated after the use of increasing concentrations of antimicrobial agents (0·25–64 μg ml−1) for the 6 isolates from three MRSA-SCCmec IV lineages in relation to the positive control (PC) without antimicrobial exposure. Filled squares, triangles and circles represent 633 (USA400/ST1), 1177 (USA800/ST5) and 943 (USA1100/ST30) biofilm producer isolates; open squares, circles and triangles represent 915 (US400/ST1) and 1314 (USA1100/ST30) nonbiofilm producer isolates, and 1112 (USA800/ST5) weak producer isolate. (image_n/jam12472-gra-0001.png) 633 (USA400/ST1); (image_n/jam12472-gra-0002.png) 915 (US400/ST1); (image_n/jam12472-gra-0002.png) 1177 (USA800/ST5); (image_n/jam12472-gra-0004.png) 1112 (USA800/ST5); (image_n/jam12472-gra-0005.png) 943 (USA1100/ST30) and (image_n/jam12472-gra-0006.png)1314 (USA1100/ST30).

The synergism experiments showed a biomass reduction of 55% for the antimicrobial associations math formula + math formula, math formula + math formula, math formula + math formula, and math formula + math formula (Fig. 3), with results equivalent to those obtained with the drugs used alone but in higher concentrations (Fig. 4). In terms of cell viability, the synergistic combinations were also more effective when compared to the individual antimicrobial tests, with reductions of 2–3 logs when the antimicrobial combinations math formula + math formula and math formula + math formula were used (Fig. 5). Although an increased activity of some drug associations has been shown compared with drugs alone against MRSA-SCCmec IV isolates, no differences between lineages and between drugs used were found (> 0·05).

Figure 3.

Biomass (a) and cell viability (log CFU cm−2) (b) evaluated after the synergistic activity of antimicrobials at different concentrations (μg ml−1) for the six isolates from three MRSA-SCCmec IV lineages in relation to the positive control (PC) without antimicrobial exposure. G16–16 μg ml−1 of gentamicin; V4–4 μg ml−1 of vancomycin, L2–2 μg ml−1 of linezolid and R2–2 μg ml−1 of rifampicin. Grey bars represent the 633 (USA400/ST1) isolate, diagonal striped bars represent the 915 (USA400/ST1) isolate, black bars represent the 1177 (USA800/ST5) isolate, dotted bars represent the 1112 (USA800/ST5) isolate, white bars represent the 943 (USA1100/ST30) isolate, and horizontal striped bars represent the 1314 (USA1100/ST30) isolate. (image) 633 (USA400/ST1); (image_n/jam12472-gra-0008.png) 915 (US400/ST1); (image) 1177 (USA800/ST5); (image_n/jam12472-gra-0009.png) 1112 (USA800/ST5); (□) 943 (USA1100/ST30) and (image_n/jam12472-gra-0010.png)1314 (USA1100/ST30).

Figure 4.

Biomass reduction in six MRSA-SCCmec IV isolates exposed to different concentrations of antimicrobial agents applied individually or combined, in relation to the positive control (PC) without antimicrobial exposure. V4–4 μg ml−1 of vancomycin, V64–64 μg ml−1 of vancomycin, R2–2 μg ml−1 of rifampicin, R64–64 μg ml−1 of rifampicin, L2–2 μg ml−1 of linezolid, L64–64 μg ml−1 of linezolid. (image) 633 (USA400/ST1); (image_n/jam12472-gra-0008.png) 915 (US400/ST1); (image) 1177 (USA800/ST5); (image_n/jam12472-gra-0009.png) 1112 (USA800/ST5); (□) 943 (USA1100/ST30) and (image_n/jam12472-gra-0010.png)1314 (USA1100/ST30).

Figure 5.

Cell viability (CFU cm−2) reduction in six MRSA-SCCmec IV isolates exposed to different concentrations of antimicrobial agents applied individually or combined, in relation to the positive control (PC) without antimicrobial exposure. V4–4 μg ml−1 of vancomycin, V64–64 μg ml−1 of vancomycin, R2–2 μg ml−1 of rifampicin, R64–64 μg ml−1 of rifampicin, L2–2 μg ml−1 of linezolid, L64–64 μg ml−1 of linezolid. (image) 633 (USA400/ST1); (image_n/jam12472-gra-0008.png) 915 (US400/ST1); (image) 1177 (USA800/ST5); (image_n/jam12472-gra-0009.png) 1112 (USA800/ST5); (□) 943 (USA1100/ST30) and (image_n/jam12472-gra-0010.png)1314 (USA1100/ST30).

Discussion

Only a few studies have evaluated the action of different antimicrobials against MRSA isolates, including those carrying SCCmec IV (Silva-Carvalho et al. 2009; Schuenck et al. 2012). However, there are no studies that have evaluated the combined action of antimicrobial agents against certain MRSA-SCCmec IV lineages, which are nowadays emerging in hospitals worldwide. In this study, for the first time, the antimicrobial susceptibility of biofilm/biomass produced by type IV lineages of MRSA that are becoming prevalent in hospitals in Rio de Janeiro was evaluated.

Initially, our survey on biofilm formation found that the different lineages of MRSA-SCCmec IV evaluated were not strong biofilm producers. These results correspond to studies of other authors that have shown that the majority of type IV isolates are nonbiofilm producers (Cha et al. 2011; Schuenck et al. 2012). Cha et al. (2011) analysed 50 MRSA-type IV isolates and classified 86% as nonbiofilm and weak biofilm producers. In a previous study by our group, Schuenck et al. (2012) analysed 28 MRSA isolates from an orthopaedic hospital and classified all the type IV isolates (STs 1, 5 and 30) as weak or moderate biofilm producers, whereas only the isolates from the Brazilian clone/type III were considered to be strong biofilm producers. In fact, according to Kaito et al. (2011), MRSA-SCCmec IV isolates do not present a psm-mec region in the mec cassette, which is related to a higher capacity of biofilm formation. Additionally, in the present study, the ST5 isolates showed a greater tendency to be biofilm producers (eight isolates/n = 14) than the isolates of ST1 lineage (1/9). This fact may be related to the origin of this lineage (paediatric clone), indicating that these isolates are more adapted to clinical environments. However, further studies involving more MRSA isolates from this and other lineages and other regions are necessary.

It is known that Staph. aureus has a great capacity to bind to different surfaces, including plastics, maintaining colonization through various forms of adhesins (Otto 2012). Thus, it was possible to evaluate the biomass formed and the number of bacterial cells present there. Furthermore, the action of the antimicrobials against the biofilms/biomass was similar to all isolates, irrespective of their ability to produce or not biofilm. Linezolid and rifampicin antibiotics were observed to be better for reducing biomass than gentamicin or vancomycin ones. Saginur et al. (2006) evaluated the biofilm antimicrobial susceptibility of 12 MRSA isolates and found that rifampicin was the single most active agent. Fernández-Barat et al. (2012) evaluated the effects of systemic treatment with linezolid compared with vancomycin on biofilm formation, in mechanically ventilated pigs with severe MRSA-induced pneumonia. They observed that the lowest bacterial burden was found in endotracheal tubes treated with linezolid in comparison with the untreated endotracheal tubes or with those treated with vancomycin, confirming the findings of the present study.

The observations in the present study concerning the limited activity of vancomycin against staphylococcal cells embedded in biofilm/biomass are consistent with other studies. (Raad et al. 2007; Wells et al. 2011). Wells et al. (2011) evaluated the viability of staphylococcal cells in mechanically dispersed biofilms and biomass formation after treatment with vancomycin; in two biofilm producer isolates cultivated in silk suture, a significant reduction in cells viability was observed only at high concentrations of the drug. However, no significant biomass reduction was observed, even in concentrations of vancomycin >20 μg ml−1. According to Wells et al. (2011), some factors may contribute to differences in the antimicrobial efficacy against biofilms/biomass, such as differences in the penetration levels of the antimicrobials into the biofilm/biomass, reduced bacterial growth rate and/or increased expression of resistance genes.

In the present study, the synergic activity of antimicrobials was more effective in reducing both biomass and number of viable cells than the drugs alone. Rifampicin (2 μg ml−1) plus linezolid (2 μg ml−1) was one of the most effective combinations, demonstrating an enhanced antibacterial effect when compared to monotherapy. According to a previous study, rifampicin is a constituent of all the combinations that are active against MRSA and is included in antibiotic therapy directed against biofilms/biomass formed by these organisms (Saginur et al. 2006). When the combination effect of oral antibiotics was evaluated for 33 biofilm-embedded MRSA isolates, rifampicin plus linezolid had a better synergism than other antimicrobial combinations (Wu et al. 2012), which was verified in the present study. Raad et al. (2007) demonstrated that rifampicin was highly active when associated with other antibiotics, particularly linezolid, in eradicating MRSA colonization on silicone discs. Also, Vergidis et al. (2011) demonstrated that combination treatment with linezolid plus rifampin or vancomycin plus rifampin was effective in an animal model of MRSA foreign body osteomyelitis in the context of retention of the infected foreign body. Although no significant differences have been found, this antimicrobial combination presented P values close to 0·05 in relation to the reduction in biomass and to the viable cell number when compared with other drug combinations. However, the reduced number of MRSA isolates evaluated may have led to these findings, and thus, further investigations involving more isolates are necessary to confirm these results.

In our study, another synergism that was observed to present a good activity against MRSA isolates was rifampicin (2 μg ml−1) plus vancomycin (4 μg ml−1). Reiter et al. (2012) studied the activity of rifampicin, individually and in combination with vancomycin, against biofilm producer isolates of MRSA. They found greater inhibition of bacterial growth after combined drug use than after use of each drug individually. However, the authors commented that the noneradication of the biofilm/biomass might contribute to bacterial persistence. Silva et al. (2011) evaluated the synergic potential of subminimum inhibitory concentrations of rifampicin plus vancomycin against clinical isolates of MRSA and coagulase-negative Staphylococcus. However, the authors observed a satisfactory synergistic effect in only two and three of the 22 isolates that were evaluated, respectively.

In the present study, the combination between linezolid and vancomycin against staphylococci biofilm/biomass was not satisfactory, especially the ineffective reduction in cell viability. Singh et al. (2009) observed that no synergistic activity was seen when these two antimicrobial agents were combined, in vitro, against five MRSA isolates from bloodstream infections.

Microscopic analysis showed similar results to the in vitro susceptibility tests, that is, only after exposure to drug combinations, a decrease in the number of viable cells was verified, as already observed by other authors (Cha et al. 2011).

Schuenck et al. (2012) showed that all isolates of MRSA were positive for the presence of the icaA gene, confirming the findings of the present study. The sasG gene was investigated by Rasmussen et al. (2013) who found a correlation between invasive Staph. aureus isolates and the presence of this gene. It is possible that the polysaccharide intercellular adhesin (PIA), essential substance in biofilm adhesion step, as well as, surface proteins of staphylococci, such as SasG present in ica-negative staphylococcal biofilms (Geoghegan et al. 2010), have some participation in biomass formation and thus contribute to the connection between cells on the material surface.

In summary, this study showed that MRSA-SCCmec IV isolates are, in general, weak or nonbiofilm producers and lineages belonging to STs 1, 5 and 30, irrespective of biofilm production, presented biomass reduction after exposure to different antimicrobials. Furthermore, antimicrobial synergisms involving math formula + math formula and math formula + math formula appear to be good therapy choices, as both combinations produced greater reductions in biomass and the number of viable staphylococcal cells. Therefore, these synergistic drug combinations might be considered in the chemotherapy of MRSA-SCCmec IV infections.

Acknowledgements

This study was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Fundação Universitária José Bonifácio (FUJB) and Programa de Núcleos de Excelência (PRONEX). The financial support through the projects: PTDC/SAUSAP/113196/2009/ FCOMP-01-0124-FEDER-016012; PEst-OE/EQB/LA0023/2013; ‘BioHealth-Biotechnology and Bioengineering approaches to improve health quality’, NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte, QREN; RECI/BBB-EBI/0179/2012/FCOMP-01-0124-FEDER-027462.

Conflict of interest

The authors declare there are no conflict of interests.

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